Isolated polypeptides, polynucleotides encoding same, transgenic plants expressing same and methods of using same

ABSTRACT

Nucleic acid constructs are provided. These constructs comprise any of the, nucleic acid sequences at least 85% identical to nucleotide sequences selected from the group consisting of SEQ ID NOs: 68, 1, 4, 5, 8, 9, 11, 13, 16, 19, 20, 23, 24, 27, 30, 32, 37, 42, 49, 50, 51, 53, 54, 55, 56, 57, 58, 64, 69, 70, 73, 77, 78, 79, 80, 84, 86, 87, 93, 94, 98, 101, 102, 103, 104, 105, 106, 107, 108 and 109 and a promoter sequence capable of directing transcription of said nucleic acid sequence in a host cell. Also provided are transgenic plants expressing these nucleic acid constructs and methods of using same.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/083,978 filed on Apr. 23, 2008, which is a National Phase of PCTPatent Application No. PCT/IL2006/001223 having International filingdate of Oct. 24, 2006, which claims the benefit of priority of U.S.Provisional Patent Application No. 60/729,181 filed on Oct. 24, 2005.

U.S. patent application Ser. No. 12/083,978 is also aContinuation-In-Part (CIP) of U.S. patent application Ser. No.11/629,411 filed on Dec. 13, 2006, now U.S. Pat. No. 7,812,218, which isa National Phase of PCT Patent Application No. PCT/IL2005/000627 havingInternational filing date of Jun. 14, 2005, which claims the benefit ofpriority of U.S. Provisional Patent Application No. 60/578,833 filed onJun. 14, 2004.

The contents of the above applications are all incorporated herein byreference.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 60793SequenceListing.txt, created on Jauary 5,2015, comprising 2,974,287 bytes, submitted concurrently with the filingof this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to isolated polypeptides, polynucleotidesencoding same, transgenic plants expressing same and methods of usingsame. Specifically the present invention can be used to increasefertilizer use efficiency and stress resistance as well as biomass,vigor and yield of transgenic plants.

Fertilizers are the fuel behind the “green revolution”, directlyresponsible for the exceptional increase in crop yields during the last40 years. The dramatic rise in crop yields could never have occurredwithout a parallel increase in fertilizer use. However, in recent yearsthere has been a growing concern with the environmental impact offertilizer use, particularly nitrogen fertilizers, on water andatmospheric pollution. Limits on fertilizer use have been legislated inseveral countries, and further restrictions are expected in the future.Greater use of fertilizers will be necessary in the future to supportfood and fiber production for rapid population growth on limited landresources.

Fertilizer is often mentioned as the number one overhead expense inagriculture. Of the three macronutrients provided as main fertilizers[Nitrogen (N), Phosphate (P) and Potassium (K)], nitrogen is the onlyone that usually needs to be replenished every year, particularly forcereals, which comprise more than half of the cultivated areasworldwide.

A common approach to promoting plant growth has been, and continues tobe, the use of nutrients (fertilizers), natural as well as synthetic.Synthetic nutrients usually provide a macronutrient in a plant-usableform, such as urea for example, and/or inorganic nitrates, phosphates,or the like compounds. While such nutrients may be applied, more orless, at the convenience of the farmer, and may be applied as often asdeemed desirable, the overuse of synthetic nutrients and the inefficientuse of synthetic nutrients are major factors responsible forenvironmental problems such as eutrophication of groundwater, nitratepollution, phosphate pollution, and the like.

Nitrogen is an essential macronutrient for the plant, responsible forbiosynthesis of amino and nucleic acids, prosthetic groups, planthormones, plant chemical defenses, etc. Nitrogen is often therate-limiting element in plant growth and all field crops have afundamental dependence on inorganic nitrogenous fertilizer. Sincefertilizer is rapidly depleted from most soil types, it must be suppliedto growing crops two or three times during the growing season.Nitrogenous fertilizer, which is usually supplied as ammonium nitrate,potassium nitrate, or urea, typically accounts for 40% of the costsassociated with crops such as corn and wheat. It has been estimated thatby 2050, more than 150 million tons of nitrogenous fertilizer will usedworldwide annually. Increased use efficiency of nitrogen by plantsshould enable crops to be cultivated with lower fertilizer input, oralternatively on soils of poorer quality and would therefore havesignificant economic impact in both developed and developingagricultural systems. An overview of the undesirable effects of nitrogenfertilizer is presented by Byrnes, Fertilizer Research, 26, pp. 209-215(1990). Although plants are able to take up organic nitrogen from theenvironment, the major part of the nitrogen utilized comes usually fromthe uptake of inorganic nitrogen in the form of ammonium (NH₄ ⁺) andnitrate (NO₃ ⁻) and its later conversion to organic nitrogen in aprocess known as assimilation.

The Nitrogen assimilation process begins with NO₃ ⁻ being converted toNH₄ ⁺ to sequentially by the enzymes Nitrate Reductase (NR) and NitriteReductase (NiR). The nitrogen is then incorporated into Glutamate (Glu)by Glutamine Synthase (GS) to obtain Glutamine (Gln). The major pathwayof nitrogen assimilation is the GS/GOGAT cycle (GlutamineSynthase/Glutamate-Oxoglutarate Amine Transferase). The remaining aminoacids are synthesized from Gln, Glu and Asn by transamination.

Nitrogen (as amino acids or in the form of nitrates) is translocated tothe shoot, where it is stored in the leaves and stalk during the rapidstep of plant development and up until flowering. In corn for example,plants accumulate the bulk of their organic nitrogen during the periodof grain germination, and until flowering. Once fertilization of theplant has occurred, grains begin to form and become the main sink ofplant Nitrogen. The stored Nitrogen can then be redistributed from theleaves and stalk that served as storage compartments until grainformation.

There are three main parameters of efficiency used to define plantNitrogen metabolism:

Nitrogen-uptake efficiency: is the amount of N in above-ground biomass(gr Nt) divided by the amount of N applied (gr/hectare);

Nitrogen utilization efficiency: is the Grain Yield (gr/plant) dividedby the amount of N in above-ground biomass (gr Nt); and

Nitrogen-use efficiency: is the Grain Yield (gr/plant) divided by theamount of N applied (gr/Ha).

The Nitrogen-uptake efficiency [the amount of N in above ground biomass(gr Nt)/N applied (gr/hectare)] is the total amount of nitrogenincorporated by the plant and is a function of the “uptake” (the plant'stransport capacity), the metabolic efficiency of the assimilationprocess and the rate of plant size development, since the mass of stalkand leaves created during growth are the actual Nitrogen-storage organs.The fraction of the assimilated Nitrogen found in a shoot that isultimately transferred to the grain (yield) is controlled enzymatically,and thus a potential site for transgenic manipulation. This parameteris, in effect, equal to the Nitrogen Utilization efficiency (NUE).Better grain-to-shoot N-partitioning most likely will improve yield andprotein content of the grain.

Similarly, the same calculations of use and utilization efficiencies canbe made for other macronutrients such as Phosphorous (P) and Potassium(K), which have a direct correlation with yield and general planttolerance.

The NUE for the main crops ranges from 30-70% only, having a directnegative impact on input expenses for the farmer, due to the excessfertilizer applied, which quickly becomes an ecological burden. Thus,nitrate-containing wastes represent an environmental problem of globalsignificance. Nitrate seepage in water causes eutrophication of lakes,rivers and seas (waters endangered because of algae growth that leads tohypoxia and destruction of marine fauna). Nitrate contamination indrinking water can cause methemoglobinemia, which is especiallydetrimental to infants and nursing mothers. In fact, the FarmingIndustry is considered as the largest nitrate polluter of surface andcoastal waters and drinking water supplies.

Genetic improvement of Fertilizer Use Efficiency (FUE) in plants can begenerated either via traditional breeding or via genetic engineering.However, to date, neither transgenic products nor classically bredenhanced FUE material have been released for commercial use. Among thereasons for this, the most important is that breeders select their elitelines under the most favorable fertilizer conditions, thus overlookingimprovements in FUE (yield being the main driver of sales and notreduction in the input costs). Attempts at transgenic solutions forimproved FUE are being carried out by companies such as Monsanto (see,for example, US Patent Applications 20020046419 to Choo, et al.; U.S.Pat. Appl. 2005010879 to Edgerton et al.; and U.S. Pat. Appl. 20060179511 to Chomet et al), Arcadia Biosciences and Biogemma.

Recently, a review summarizing attempts to improve FUE by transgenicmeans that have been undertaken by academic laboratories was published(Good A G et al. Trends Plant Sci. 2004 December; 9(12):597-605).Encouraging results were reported by Yanagisawa and coworkers (Proc NatlAcad Sci USA. 2004 May 18; 101(20):7833-8) who found that a geneticallyengineered increase in carbon skeleton production (2-Oxoglutarate, OGfrom the GS/GOGAT cycle) sustained growth of transgenic Arabidopsisunder low nitrogen conditions. As many enzymes are involved in carbonskeleton production, the transgene was a key transcriptional factor(Dof1) that activated multiple genes involved in the pathway. Nitrogencontent was higher in the Arabidopsis transgenic plants by approximately30% under low nitrogen conditions. U.S. Pat. No. 6,084,153 to Good etal. discloses the use of a stress responsive promoter to control theexpression of Alanine Amine Transferase (AlaAT). Good et al. furtherdisclosed that transgenic canola plants improved drought and nitrogendeficiency tolerance when compared to control plants. However, neitherthe Dof1 constructs of Yanagisawa et al, nor the drought-induced AlaATconstructs of Good et al. have been evaluated in commercial lines, undertrue field conditions. Hence the economic relevance of the results isyet to be proven.

There is thus a widely recognized need for, and it would be highlyadvantageous to identify polynucleotides and polypeptides which improvefertilizer use/uptake efficiency in transgenic plants expressing same,which are devoid of the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anucleic acid construct comprising a nucleic acid sequence at least 85%identical to a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 68, 1, 4, 5, 8, 9, 11, 13, 16, 19, 20, 23, 24, 27, 30, 32,37, 42, 49, 50, 51, 53, 54, 55, 56, 57, 58, 64, 69, 70, 73, 77, 78, 79,80, 84, 86, 87, 93, 94, 98, 101, 102, 103, 104, 105, 106, 107, 108 and109 and a promoter sequence capable of directing transcription of thenucleic acid sequence in a host cell.

According to still further features in the described preferredembodiments the nucleic acid sequence is as set forth in SEQ ID NO: 68,1, 4, 5, 8, 9, 11, 13, 16, 19, 20, 23, 24, 27, 30, 32, 37, 42, 49, 50,51, 53, 54, 55, 56, 57, 58, 64, 69, 70, 73, 77, 78, 79, 80, 84, 86, 87,93, 94, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 219-767, 1317,1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329,1330, 1331, 1332, 1333, 1334, 1335 or 1336.

According to another aspect of the present invention there is providedan isolated polypeptide, comprising an amino acid sequence at least 85%homologous to the amino acid sequence set forth in SEQ ID NO: 177, 110,113, 114, 117, 118, 120, 122, 125, 128, 129, 132, 133, 136, 139, 141,146, 151, 158, 159, 160, 162, 163, 164, 165, 166, 167, 173, 178, 179,182, 186, 187, 188, 189, 193, 195, 196, 202, 203, 207, 210, 211, 212,213, 214, 215, 216, 217 or 218.

According to yet another aspect of the present invention there isprovided a plant cell comprising an exogenous polynucleotide whichcomprises a nucleic acid sequence encoding a polypeptide having an aminoacid sequence at least 85% homologous to SEQ ID NO: 177, 110, 113, 114,117, 118, 120, 122, 125, 128, 129, 132, 133, 136, 139, 141, 146, 151,158, 159, 160, 162, 163, 164, 165, 166, 167, 173, 178, 179, 182, 186,187, 188, 189, 193, 195, 196, 202, 203, 207, 210, 211, 212, 213, 214,215, 216, 217 or 218.

According to still further features in the described preferredembodiments the plant cell forms a part of a plant.

According to still further features in the described preferredembodiments the amino acid sequence is as set forth in SEQ ID NO: 177,110, 113, 114, 117, 118, 120, 122, 125, 128, 129, 132, 133, 136, 139,141, 146, 151, 158, 159, 160, 162, 163, 164, 165, 166, 167, 173, 178,179, 182, 186, 187, 188, 189, 193, 195, 196, 202, 203, 207, 210, 211,212, 213, 214, 215, 216, 217, 218, 768-1051, 1053-1098, 1100-1315 or1316.

According to still another aspect of the present invention there isprovided a method of increasing tolerance of a plant to a stresscondition, comprising expressing within the plant an exogenouspolynucleotide encoding a polypeptide having an amino acid sequence atleast 85% homologous to SEQ ID NO: 177, 110, 113, 114, 117, 118, 120,122, 125, 128, 129, 132, 133, 136, 139, 141, 146, 151, 158, 159, 160,162, 163, 164, 165, 166, 167, 173, 178, 179, 182, 186, 187, 188, 189,193, 195, 196, 202, 203, 207, 210, 211, 212, 213, 214, 215, 216, 217,218, 1341 or 1343, thereby increasing the tolerance of the plant to thestress condition.

According to an additional aspect of the present invention there isprovided a method of increasing biomass, vigor and/or yield of a plant,comprising expressing within the plant an exogenous polynucleotideencoding a polypeptide having an amino acid sequence at least 85%homologous to SEQ ID NO: 177, 110, 113, 114, 117, 118, 120, 122, 125,128, 129, 132, 133, 136, 139, 141, 146, 151, 158, 159, 160, 162, 163,164, 165, 166, 167, 173, 178, 179, 182, 186, 187, 188, 189, 193, 195,196, 202, 203, 207, 210, 211, 212, 213, 214, 215, 216, 217, 218, 1341 or1343, thereby increasing biomass, vigor and/or yield of the plant.

According to yet an additional aspect of the present invention there isprovided a method of increasing fertilizer use efficiency and/or uptakeof a plant comprising expressing within the plant an exogenouspolynucleotide encoding a polypeptide having an amino acid sequence atleast 85% homologous to SEQ ID NO: 177, 110, 113, 114, 117, 118, 120,122, 125, 128, 129, 132, 133, 136, 139, 141, 146, 151, 158, 159, 160,162, 163, 164, 165, 166, 167, 173, 178, 179, 182, 186, 187, 188, 189,193, 195, 196, 202, 203, 207, 210, 211, 212, 213, 214, 215, 216, 217,218, 1341 or 1343, thereby increasing fertilizer use efficiency and/oruptake of the plant.

According to still further features in the described preferredembodiments the expressing is effected by:

-   -   (a) transforming a cell of the plant with the exogenous        polynucleotide;    -   (b) generating a mature plant from the cell; and    -   (c) cultivating the mature plant under conditions suitable for        expressing the exogenous polynucleotide within the mature plant.

According to still further features in the described preferredembodiments the transforming is effected by introducing to the plantcell a nucleic acid construct including the exogenous polynucleotide andat least one promoter capable of directing transcription of theexogenous polynucleotide in the plant cell.

According to still further features in the described preferredembodiments the at least one promoter is a constitutive promoter.

According to further features in preferred embodiments of the inventiondescribed below, the promoter is a constitutive promoter.

According to still further features in the described preferredembodiments the constitutive promoter is CaMV 35S promoter.

According to still further features in the described preferredembodiments the promoter is an inducible promoter.

According to still further features in the described preferredembodiments the inducible promoter is an abiotic stress induciblepromoter.

According to still further features in the described preferredembodiments the promoter is a tissue specific promoter.

According to still further features in the described preferredembodiments the tissue is a root tissue.

According to still further features in the described preferredembodiments the host cell is a plant cell.

According to still further features in the described preferredembodiments the plant cell forms a part of a dicotyledonous plant cell.

According to still further features in the described preferredembodiments the plant cell forms a part of a monocotyledonous plantcell.

According to still further features in the described preferredembodiments the stress condition is an abiotic stress.

According to still further features in the described preferredembodiments the abiotic stress is selected from the group consisting ofsalinity, drought, low temperature, high temperature, heavy metaltoxicity, anaerobiosis, osmoticum, and nutrient deficiency.

According to still further features in the described preferredembodiments the nutrient is nitrogen.

According to still further features in the described preferredembodiments the plant is a dicotyledonous plant.

According to still further features in the described preferredembodiments the plant is a monocotyledonous plant.

According to still further features in the described preferredembodiments the polynucleotide comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 68, 1, 4, 5, 8, 9, 11,13, 16, 19, 20, 23, 24, 27, 30, 32, 37, 42, 49, 50, 51, 53, 54, 55, 56,57, 58, 64, 69, 70, 73, 77, 78, 79, 80, 84, 86, 87, 93, 94, 98, 101,102, 103, 104, 105, 106, 107, 108, 109, 219-767, 1317, 1318, 1319, 1320,1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332,1333, 1334, 1335, 1336, 1340 or 1342.

According to still further features in the described preferredembodiments the amino acid sequence is as set forth in SEQ ID NO: 177,110, 113, 114, 117, 118, 120, 122, 125, 128, 129, 132, 133, 136, 139,141, 146, 151, 158, 159, 160, 162, 163, 164, 165, 166, 167, 173, 178,179, 182, 186, 187, 188, 189, 193, 195, 196, 202, 203, 207, 210, 211,212, 213, 214, 215, 216, 217, 218, 768-1051, 1053-1098, 1100-1316, 1341or 1343.

According to still further features in the described preferredembodiments the conditions are abiotic stress conditions.

According to still further features in the described preferredembodiments the conditions are fertilizer deficiency conditions.

According to still an additional aspect of the present invention thereis provided a method for irrigation comprising:

-   -   (a) placing on or in a soil a dripping irrigation system so as        to obtain irrigation holes distributed 20-40 cm from one another        in the X and Y directions; and    -   (b) continuously irrigating through each of said irrigation        holes at an irrigation rate of 0.5-2 liter water per hour.

According to a further aspect of the present invention there is providedan irrigation system for mimicking drought conditions comprising:

-   -   (i) a dripping irrigation system having irrigation holes        distributed 20-40 cm from one another in the X and Y directions;        and    -   (ii) a water supply and control system for continuously        irrigating through each of said irrigation holes 0.5-2 liter        water per hour.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing polynucleotides andpolypeptides which improve fertilizer use/uptake efficiency intransgenic plants expressing same.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1A is a schematic illustration describing the development of theplant root system in drought. Scarce soil humidity induces the formationof a deeper root system. The drawing is adapted from J E Weaver [RootDevelopment of Field Crops. McGraw Hill Inc., New York, 291 p. (1926)].

FIG. 1B is a schematic representation of the biological strategieschosen to improve the NUE trait and the translation of the strategies tothe queries performed during computational data mining.

FIGS. 2A-D show digital expression of FUE_1.

FIGS. 3A-D are graphs showing expression of control genes (Nitrogentransporters and Glutamine synthase) as a function of nitrogen contentin the soil. Plants were grown in 10 liter pots with increasingconcentrations of either NH₄NO₃ (at 0.005, 0.05, 0.5 and 5 mM) or KNO₃(at 0.01, 0.1, 1 or 5 mM). Plants were grown for approximately 30 daysand tissues were snap frozen in liquid nitrogen. RNA was extracted fromthe tissues and later treated with DNAse. RNA was reverse-transcribedand used for quantification assays using Real Time PCR. Fornormalization, the expression at each point was divided by the geometricmean of the expression of four housekeeping as described in Example 2.The results shown are the ratio between the normalized expression andthe expression levels measured at the highest concentration checked (5mM NH₄NO₃ or 5 mM KNO₃).

FIGS. 4A-K are graphs showing correlated expression of polynucleotidesequences of the present invention with nitrogen availability.

FIGS. 5A-E are graphs showing inversely correlated expression ofpolynucleotide sequences of the present invention with nitrogenavailability.

FIG. 6 is a schematic stepwise representation of the technology used forplant rosette area quantitation from digital images. The processing stepfilters the green parts of the individual plants. After designation ofindividual ROIs (Region of Interest) the area covered by the rosettearea is calculated and exported to a worksheet.

FIGS. 7A-B are photographs showing the ability of polynucleotides of thepresent invention to increase nitrogen use efficiency. FIG. 7Atransgenic—T1 plants expressing FUE_504 (SEQ ID NO: 108) under the TT105promoter. FIG. 7B one representative transgenic event expressing FUE_43(SEQ ID NO: 70) under the 35S promoter. Plants were grown at verylimiting nitrogen conditions (0.05 and 0.2 mM combined inorganicnitrogen). Control plants (expressing a reporter gene under the samepromoter or non-transgenic plants) were treated similarly. The notoriousdifferences in plantlet size are translated into significant differencesin the measured fresh weight of the plantlets.

FIG. 8 is a photograph showing transgenic plants from three independenttransgenic events expressing FUE_34_Evo (SEQ ID NO: 54). The plant showsimpressive branching at every node as determined by a triple blindassay. The root system from the plants was also highly branched andcompact (not shown).

FIG. 9 shows five independent transgenic events of plants expressingFUE_40 (also termed FUE6/40, SEQ ID NO: 11). It is clearly notable thatthe flowering stem is unusually upright and stiff. In addition, anunusually high number of siliques can be counted. As a result,transgenic plants expressing FUE_40 are remarkably prolific.

FIG. 10 a scheme of an irrigation system in accordance with theteachings of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of isolated polypeptides, polynucleotidesencoding same, transgenic plants expressing same and methods of usingsame. Specifically the present invention can be used to increasefertilizer use efficiency and stress resistance as well as biomass,vigor and yield of transgenic plants.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

As the cost of Nitrogen (N) fertilizer increases and profit of cropplants production is put as risk, scientists are challenged to developstrategies that improve N use efficiency (NUE). Thus, under orover-fertilization results in a loss of profit. Lower rates of N appliedresult in reduced biomass as may be evidenced by reduced tillering,size, poor grain fill, reduced yields and low amino acid and proteincontent. Alternatively, over application of nitrogen can result inreduced yield, higher input cost and increased risk to the environment.

While reducing the present invention to practice, the present inventorsuncovered through laborious bioinformatic analyses and experimentation,polynucleotide sequences and polypeptides encoded therefrom which can beused to generate transgenic plants with improved fertilizer useefficiency, stress tolerance, nutritional value (e.g., amino acids andprotein content), biomass, yield and/or vigor.

As is illustrated hereinbelow and in the examples section which follows,maize polynucleotide sequences were selected based on several criteria(see Example 1 of the Examples section which follows). These includedhigh levels of calculated digital expression at the root tips especiallyin water stressed roots (e.g., drought). A secondary filter for theabove polynucleotide sequences was based on the digital expression oftheir orthologous sequences (i.e., dicotyledon plants or othermonocotyledons species). Orthologous genes showing similar digitalexpression as the maize genes (e.g., root libraries of stress treatedplants subjected) were selected. Data mining and annotation tools wereused to filter genes which may have broad effects on cell metabolism.For example genes which can modify root architecture, increase nitrogenstorage capacity, improve nitrogen assimilation process, and enhance thestay-green trait were selected. Those genes were finally selected formolecular validation (see Examples 3 and 9). A schematic representationof the computational filters applied to identify the polynucleotidesequences of the present invention is presented in FIGS. 1B and 1 t isprovided for illustration only.

Sequences thus identified were experimentally validated. As shown inExamples 6-11 transgenic plants expressing the nucleic acid sequences ofthe present invention were shown to have increased fertilizer use/uptakeefficiency, tolerance to abiotic stress, biomass and yield. Theseresults strongly support the robustness of the methodology of thepresent invention and substantiate the use of these genes inagriculture.

Thus, according to one aspect of the present invention, there isprovided a nucleic acid construct comprising a nucleic acid sequence atleast about 70%, at least about 75%, at least about 80%, at least about81%, at least about 82%, at least about 83%, at least about 84%, atleast about 85%, at least about 86%, at least about 87%, at least about88%, at least about 89%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or more say 100% identical to anucleotide sequence selected from the group consisting of SEQ ID NO: 68,1, 4, 5, 8, 9, 11, 13, 16, 19, 20, 23, 24, 27, 30, 32, 37, 42, 49, 50,51, 53, 54, 55, 56, 57, 58, 64, 69, 70, 73, 77, 78, 79, 80, 84, 86, 87,93, 94, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1340 and 1342.

Nucleic acid sequences may encode polypeptide sequences comprising anamino acid sequence at least about 70%, at least about 75%, at leastabout 80%, at least about 81%, at least about 82%, at least about 83%,at least about 84%, at least about 85%, at least about 86%, at leastabout 87%, at least about 88%, at least about 89%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or more say100% homologous to SEQ ID NO 177, 110, 113, 114, 117, 118, 120, 122,125, 128, 129, 132, 133, 136, 139, 141, 146, 151, 158, 159, 160, 162,163, 164, 165, 166, 167, 173, 178, 179, 182, 186, 187, 188, 189, 193,195, 196, 202, 203, 207, 210, 211, 212, 213, 214, 215, 216, 217, 218,1341 or 1343.

Homology (e.g., percent homology) can be determined using any homologycomparison software, including for example, the BlastP software of theNational Center of Biotechnology Information (NCBI) such as by usingdefault parameters.

Identity (e.g., percent homology) can be determined using any homologycomparison software, including for example, the BlastN software of theNational Center of Biotechnology Information (NCBI) such as by usingdefault parameters.

According to one preferred embodiment of this aspect of the presentinvention the isolated polynucleotide is as set forth is SEQ ID NO: 68,1, 4, 5, 8, 9, 11, 13, 16, 19, 20, 23, 24, 27, 30, 32, 37, 42, 49, 50,51, 53, 54, 55, 56, 57, 58, 64, 69, 70, 73, 77, 78, 79, 80, 84, 86, 87,93, 94, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 219-767, 1317,1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329,1330, 1331, 1332, 1333, 1334, 1335 or 1336.

A nucleic acid sequence (also termed herein as isolated polynucleotide)of the present invention refers to a single or double stranded nucleicacid sequence which is isolated and provided in the form of an RNAsequence, a complementary polynucleotide sequence (cDNA), a genomicpolynucleotide sequence and/or a composite polynucleotide sequences(e.g., a combination of the above).

As used herein the phrase “complementary polynucleotide sequence” refersto a sequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.Such a sequence can be subsequently amplified in vivo or in vitro usinga DNA dependent DNA polymerase.

As used herein the phrase “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” refers toa sequence, which is at least partially complementary and at leastpartially genomic. A composite sequence can include some exonalsequences required to encode the polypeptide of the present invention,as well as some intronic sequences interposing therebetween. Theintronic sequences can be of any source, including of other genes, andtypically will include conserved splicing signal sequences. Suchintronic sequences may further include cis acting expression regulatoryelements.

Nucleic acid sequences of the polypeptides of the present invention maybe optimized for plant expression. Such optimized sequences are providedin SEQ ID NOs: 1317, 1319, 1320, 1321, 1322, 1324, 1325, 1326, 1327,1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335 and 1336. Examples ofsuch sequence modifications include, but are not limited to, an alteredG/C content to more closely approach that typically found in the plantspecies of interest, and the removal of codons atypically found in theplant species commonly referred to as codon optimization.

The phrase “codon optimization” refers to the selection of appropriateDNA nucleotides for use within a structural gene or fragment thereofthat approaches codon usage within the plant of interest. Therefore, anoptimized gene or nucleic acid sequence refers to a gene in which thenucleotide sequence of a native or naturally occurring gene has beenmodified in order to utilize statistically-preferred orstatistically-favored codons within the plant. The nucleotide sequencetypically is examined at the DNA level and the coding region optimizedfor expression in the plant species determined using any suitableprocedure, for example as described in Sardana et al. (1996, Plant CellReports 15:677-681). In this method, the standard deviation of codonusage, a measure of codon usage bias, may be calculated by first findingthe squared proportional deviation of usage of each codon of the nativegene relative to that of highly expressed plant genes, followed by acalculation of the average squared deviation. The formula used is: 1SDCU=n=1 N [(Xn−Yn)/Yn]2/N, where Xn refers to the frequency of usage ofcodon n in highly expressed plant genes, where Yn to the frequency ofusage of codon n in the gene of interest and N refers to the totalnumber of codons in the gene of interest. A table of codon usage fromhighly expressed genes of dicotyledonous plants is compiled using thedata of Murray et al. (1989, Nuc Acids Res. 17:477-498).

One method of optimizing the nucleic acid sequence in accordance withthe preferred codon usage for a particular plant cell type is based onthe direct use, without performing any extra statistical calculations,of codon optimization tables such as those provided on-line at the CodonUsage Database through the NIAS (National Institute of AgrobiologicalSciences) DNA bank in Japan (Hypertext Transfer Protocol://World WideWeb (dot) kazusa (dot) or (dot) jp/codon/). The Codon Usage Databasecontains codon usage tables for a number of different species, with eachcodon usage table having been statistically determined based on the datapresent in Genbank.

By using the above tables to determine the most preferred or mostfavored codons for each amino acid in a particular species (for example,rice), a naturally-occurring nucleotide sequence encoding a protein ofinterest can be codon optimized for that particular plant species. Thisis effected by replacing codons that may have a low statisticalincidence in the particular species genome with corresponding codons, inregard to an amino acid, that are statistically more favored. However,one or more less-favored codons may be selected to delete existingrestriction sites, to create new ones at potentially useful junctions(5′ and 3′ ends to add signal peptide or termination cassettes, internalsites that might be used to cut and splice segments together to producea correct full-length sequence), or to eliminate nucleotide sequencesthat may negatively effect mRNA stability or expression.

The naturally-occurring encoding nucleotide sequence may already, inadvance of any modification, contain a number of codons that correspondto a statistically-favored codon in a particular plant species.Therefore, codon optimization of the native nucleotide sequence maycomprise determining which codons, within the native nucleotidesequence, are not statistically-favored with regards to a particularplant, and modifying these codons in accordance with a codon usage tableof the particular plant to produce a codon optimized derivative. Amodified nucleotide sequence may be fully or partially optimized forplant codon usage provided that the protein encoded by the modifiednucleotide sequence is produced at a level higher than the proteinencoded by the corresponding naturally occurring or native gene.Construction of synthetic genes by altering the codon usage is describedin for example PCT Patent Application 93/07278.

Thus, the present invention encompasses nucleic acid sequences describedhereinabove; fragments thereof, sequences hybridizable therewith,sequences homologous thereto, sequences orthologous thereto, sequencesencoding similar polypeptides with different codon usage, alteredsequences characterized by mutations, such as deletion, insertion orsubstitution of one or more nucleotides, either naturally occurring orman induced, either randomly or in a targeted fashion.

Nucleic acid sequences of the present invention may encode previouslyuncharacterized polypeptides

Thus the present invention provides a polypeptide having an amino acidsequence at least about 70%, at least about 75%, at least about 80%, atleast about 81%, at least about 82%, at least about 83%, at least about84%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, or more say 100% homologousto an amino acid sequence selected from the group consisting of SEQ IDNO: 177, 110, 113, 114, 117, 118, 120, 122, 125, 128, 129, 132, 133,136, 139, 141, 146, 151, 158, 159, 160, 162, 163, 164, 165, 166, 167,173, 178, 179, 182, 186, 187, 188, 189, 193, 195, 196, 202, 203, 207,210, 211, 212, 213, 214, 215, 216, 217, 218, 1341 or 1343.

According to an embodiment of this aspect of the present invention theisolated polypeptide comprises an amino acid sequence selected from thegroup consisting of 177, 110, 113, 114, 117, 118, 120, 122, 125, 128,129, 132, 133, 136, 139, 141, 146, 151, 158, 159, 160, 162, 163, 164,165, 166, 167, 173, 178, 179, 182, 186, 187, 188, 189, 193, 195, 196,202, 203, 207, 210, 211, 212, 213, 214, 215, 216, 217, 218, 768-1051,1053-1098, 1100-1315, 1316, 1341 or 1343.

The present invention also encompasses sequences homologous andorthologous to the above mentioned polypeptides, fragments of the abovedescribed polypeptides and polypeptides having mutations, such asdeletions, insertions or substitutions of one or more amino acids,either naturally occurring or man induced, either randomly or in atargeted fashion.

Polynucleotides and polypeptides of the present invention are used forplant expression.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,roots (including tubers), and plant cells, tissues and organs. The term“plant” also therefore encompasses suspension cultures, embryos,meristematic regions, callus tissue, leaves, gametophytes, sporophytes,pollen, and microspores. Plants that are particularly useful in themethods of the invention include all plants which belong to thesuperfamily Viridiplantae, in particular monocotyledonous anddicotyledonous plants which are of commercial value, including a fodderor forage legume, ornamental plant, food crop, tree, or shrub selectedfrom the following non-limiting list comprising Acacia spp., Acer spp.,Actinidia spp., Aesculus spp., Agathis australis, Albizia amara,Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Asteliafragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassicaspp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadabafarinosa, Calliandra spp, Camella sinensis, Canna indica, Capsicum spp.,Cassia spp., Cent roema pubescens, Chaenomeles spp., Cinnamomum cassia,Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneasterserotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyatheadealbata, Cydonia oblonga, Ciyptomeria laponica, Cymbopogon spp.,Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davaliladivaricata, Desmodium spp., Dicksonia squarosa, Diheteropogonamplectens, Dioclea spp, Dolichos spp., Doiycnium rectum, Echinochloapyramidalis, Ehrartia spp., Eleusine coracana, Era grestis spp.,Erythrina spp., Eucalyptus spp, Euclea. schimpen Eulalia villosa,Fagopyrum spp., Felloa sellowiana, Fragaria spp., Flemingia spp,Freycinetia banksii, Geranium thunbergi, Ginkgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Gre villea spp.,Guibourtia coleosperma, Hedysarum spp., Hemarthia altissima, Heteropogoncon tortus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum,Hyperthelia dissoluta, Indigo incarnata, Iris spp., Jatropha curcas,Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaenaleucocephala, Loudetia simplex, Lotonus bainesi, Lotus spp., Macrotylomaaxifiare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoiaglyptostroboides, Musa sapientum, Nicotianum spp., Onobtychis spp.,Ornithopus spp., Oryza spp., Peltophorum african urn, Pennisetum spp.,Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis,Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisumsativum, Podocarpus totara, Pogonarthria flecki, Pogonarthria squarrosa,Populus spp., Prosopis cineraria, Pseudotsuga menziesi, Pterolobiumstellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata,Rhopalostylis sapida, Rhu.s natalensis, Ribes grossularia, Ribes spp.,Robinia pseudoacacia, Rosa spp., Rub us spp., Salix spp., Schyzachyriumsanguineurn, Sciadopitys verticillata, Sequoia sempen'irens,Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolusfimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp,Taxodium distichum, Themeda triandra, Trifollum spp., Triticum spp.,Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsoniapyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke,asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot,cauliflower, celery, collard greens, flax, kale, lentil, ollseed rape,okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane,sunflower, tomato, squash tea, trees. Alternatively algae and othernon-Viridiplantae can be used.

Expressing the exogenous polynucleotide of the present invention withinthe plant can be effected by transforming one or more cells of the plantwith the exogenous polynucleotide, followed by generating a mature plantfrom the transformed cells and cultivating the mature plant underconditions suitable for expressing the exogenous polynucleotide withinthe mature plant.

Preferably, the transformation is effected by introducing to the plantcell a nucleic acid construct which includes the exogenouspolynucleotide of the present invention and at least one promotercapable of directing transcription of the exogenous polynucleotide inthe plant cell. Further details of suitable transformation approachesare provided hereinbelow.

As used herein, the term “promoter” refers to a region of DNA which liesupstream of the transcriptional initiation site of a gene to which RNApolymerase binds to initiate transcription of RNA. The promoter controlswhere (e.g., which portion of a plant, which organ within an animal,etc.) and/or when (e.g., which stage or condition in the lifetime of anorganism) the gene is expressed.

Any suitable promoter sequence can be used by the nucleic acid constructof the present invention. The following types of promoters arenon-limiting examples of promoters used to over-express the selectedgenes: general promoters, root specific promoters, root-tips specificpromoters, drought-induced root promoters, biotic stress-inducedpromoters, abiotic stress-induced promoters, nitrogen induced promoters,ammonium or nitrate induced promoters, phosphate fertilizer-inducedpromoters, leaf specific promoters, inducible promoters, constitutivepromoters, promoters with two or more of the characteristics describedabove, or other novel promoters.

Choice of the promoter is based largely on the phenotype of interest andis determined by such factors as tissue (e.g., seed, fruit, root,pollen, vascular tissue, flower, carpel, etc.), inducibility (e.g., inresponse to wounding, heat, cold, drought, light, pathogens, etc.),timing, developmental stage, and the like. Numerous known promoters havebeen characterized and can favorably be employed to promote expressionof a polynucleotide of the invention in a transgenic plant or cell ofinterest. Measures should be taken, however, to select a promoter whichwill mediate desirable expression levels of the transgene so as to avoidreallocating excessive energetic resources which may affect final yield,strength, mass and lodging and incidence of foliar pathogens. Thisshould also be viewed from an economic perspective.

Suitable constitutive promoters include, for example, CaMV 35S promoter(SEQ ID NO: 1337; Odell et al., Nature 313:810-812, 1985); ArabidopsisAt6669 promoter (SEQ ID NO: 1514, PCT No WO2004/104162); TT105 (SEQ IDNO: 1339, PCT NO.: WO2004/081173) maize Ubi 1 (Christensen et al., PlantSol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588,1991); and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76,1995). Other constitutive promoters include those in U.S. Pat. Nos.5,659,026, 5,608,149; 5.608,144; 5,604,121; 5,569,597: 5,466,785;5,399,680; 5,268,463; and 5,608,142.

Suitable tissue-specific promoters include, but not limited to,leaf-specific promoters such as described, for example, by Yamamoto etal., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67,1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor etal., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol.23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA90:9586-9590, 1993. Especially preferred are root promoters such as theROOTP promoter [SEQ ID NO: 1338; Upstream region of the gene ATXTH19(AT4G30290, Xyloglucan endotransglucosylase/hydrolase 19, described inVissenberg K, et al. Plant Cell Physiol. 2005 January; 46(1):192-200].

A variety of plant gene promoters are known to regulate gene expressionin response to environmental, hormonal, chemical, developmental signals,and in a tissue-active manner. Examples of seed-specific promoters (suchas the napin, phaseolin or DC3 promoter described in U.S. Pat. No.5,773,697), fruit-specific promoters that are active during fruitripening, such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the2A11 promoter (U.S. Pat. No. 4,943,674) and the tomato polygalacturonasepromoter (Bird et al. (1988) Plant Mol. Biol. 11: 651-662),root-specific promoters, such as ARSK1, and those disclosed in U.S. Pat.Nos. 5,618,988, 5,837,848 and 5,905,186, epidermis-specific promoters,including CUT1 (Kunst et al. (1999) Biochem. Soc. Tians. 28: 651-654),pollen-active promoters such as PTA29, PTA26 and PTA 13 (U.S. Pat. No.5,792,929), promoters active in vascular tissue (Ringli and Keller(1998) Plant Mol. Biol. 37: 977-988), flower-specific (Kaiser et al.(1995) Plant Mol. Biol. 28: 231-243), pollen (Baerson et al. (1994)Plant Mol. Biol. 26: 1947-1959), carpels (Ohl et al. (1990) Plant Cell2: 837-848), pollen and ovules (Baerson et al. (1993) Plant Mol. Biol.22: 255-267), auxin-inducible promoters (such as that described in vander Kop et al. (1999) Plant Mol. Biol. 39: 979-990 or Baumann et al.(1999) Plant Cell 11: 323-334), cytokinin-inducible promoter(Guevara-Garcia (1998) Plant Mol. Biol. 38: 743-753), promotersresponsive to gibberellin (Shi et al. (1998) Plant Mol. Biol. 38:1053-1060, Willmott et al. (1998) Plant Mol. Biol. 38: 817-825) and thelike. Additional promoters are those that elicit expression in responseto heat (Ainley et al. (1993) Plant Mol. Biol. 22: 13-23), light (e.g.,the pea rbcS-3A promoter, described in Kuhlemeier et al. (1989) PlantCell 1: 471-478, and the maize rbcS promoter, described in Schaffier andSheen (1991) Plant Cell 3: 997-1012); wounding (e.g., wunl, described inSiebertz et al. (1989) Plant Cell 1: 961-968), pathogens (such as thePR-1 promoter described in Buchel et al. (1999) Plant Mol. Biol. 40:387-396, and the PDF1.2 promoter described in Manners et al. (1998)Plant Mol. Biol. 38: 1071-1080), and chemicals such as methyl jasmonateor salicylic acid (Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol.Biol. 48: 89-108). In addition, the timing of the expression can becontrolled by using promoters such as those acting at senescence (Ganand Amasino (1995) Science 270: 1986-1988); or late seed development(Odell et al. (1994) Plant Physiol. 106: 447-458).

Other examples of promoters are a SUC2 promoter (Truernit and Sauer,Planta. (1995) 196:564-70), and a stress-inducible promoter such asRD29A (Yamaguchi-Shinozaki and Shinozaki K. Plant Cell (1994)6:251-264), promoters fro the PlantProm database (Shahmuradov et al.Nucleic Acids Res. (2003) 31:114-7), the rice CatB promoter (Iwamoto etal. Plant Physiol Biochem. (2004) 42:241-9), the root specific andphosphate-deficiency inducible barley promoters of the phosphatetransporter gene family (HvPht1;1 and HvPht1;2) (Schunmann et al.(2004); 55:855-65), tissue specific and constitutive promotersillustrated in patent No: WO2004/081173, in patent No: U.S. Pat. No.5,633,363, in patent No: WO 2000/15662, in patent No: WO 2004/013169, inpatent No: US 2005/010974, in patent No: WO 2005/035770, in patent No:US 2001/0047525, in patent No: U.S. Pat. No. 5,837,848, in patent No:U.S. Pat. No. 6,018,099, etc.

The nucleic acid construct of the present invention preferably furtherincludes an appropriate selectable marker and/or an origin ofreplication. Preferably, the nucleic acid construct utilized is ashuttle vector, which can propagate both in E. coli (wherein theconstruct comprises an appropriate selectable marker and origin ofreplication) and be compatible with propagation in cells. The constructaccording to the present invention can be, for example, a plasmid, abacmid, a phagemid, a cosmid, a phage, a virus or an artificialchromosome.

The nucleic acid construct of the present invention can be utilized tostably or transiently transform plant cells. In stable transformation,the exogenous polynucleotide of the present invention is integrated intothe plant genome and as such it represents a stable and inherited trait.In transient transformation, the exogenous polynucleotide is expressedby the cell transformed but it is not integrated into the genome and assuch it represents a transient trait.

There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev.Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNAinto plant genomic DNA include two main approaches:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; glass fibers or silicon carbide whiskertransformation of cell cultures, embryos or callus tissue, U.S. Pat. No.5,464,765 or by the direct incubation of DNA with germinating pollen,DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman,G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. Horsch et al. in Plant Molecular Biology Manual A5,Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementaryapproach employs the Agrobacterium delivery system in combination withvacuum infiltration. The Agrobacterium system is especially viable inthe creation of transgenic dicotyledonous plants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, the protoplasts are briefly exposed to a strongelectric field. In microinjection, the DNA is mechanically injecteddirectly into the cells using very small micropipettes. In microparticlebombardment, the DNA is adsorbed on microprojectiles such as magnesiumsulfate crystals or tungsten particles, and the microprojectiles arephysically accelerated into cells or plant tissues.

Following stable transformation plant propagation is exercised. The mostcommon method of plant propagation is by seed. Regeneration by seedpropagation, however, has the deficiency that due to heterozygositythere is a lack of uniformity in the crop, since seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transformedplant be produced such that the regenerated plant has the identicaltraits and characteristics of the parent transgenic plant. Therefore, itis preferred that the transformed plant be regenerated bymicropropagation which provides a rapid, consistent reproduction of thetransformed plants.

Micropropagation is a process of growing new generation plants from asingle piece of tissue that has been excised from a selected parentplant or cultivar. This process permits the mass reproduction of plantshaving the preferred tissue expressing the fusion protein. The newgeneration plants which are produced are genetically identical to, andhave all of the characteristics of, the original plant. Micropropagationallows mass production of quality plant material in a short period oftime and offers a rapid multiplication of selected cultivars in thepreservation of the characteristics of the original transgenic ortransformed plant. The advantages of cloning plants are the speed ofplant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration ofculture medium or growth conditions between stages. Thus, themicropropagation process involves four basic stages: Stage one, initialtissue culturing; stage two, tissue culture multiplication; stage three,differentiation and plant formation; and stage four, greenhouseculturing and hardening. During stage one, initial tissue culturing, thetissue culture is established and certified contaminant-free. Duringstage two, the initial tissue culture is multiplied until a sufficientnumber of tissue samples are produced to meet production goals. Duringstage three, the tissue samples grown in stage two are divided and growninto individual plantlets. At stage four, the transformed plantlets aretransferred to a greenhouse for hardening where the plants' tolerance tolight is gradually increased so that it can be grown in the naturalenvironment.

Preferably, mature transformed plants generated as described above arefurther selected for the trait of interest (e.g., improved FUE, stresstolerance etc.). Examples of such screening assays are providedhereinbelow and in the Examples section which follows. Thus, forexample, transgenic plants may be screened for improved nutritionalvalue (e.g., improved oil, amino acids and/or protein content, as wellas N content per se) under normal or stress conditions as will befurther described hereinbelow. Alternatively or additionally,transformed and non-transformed (wild type) plants are exposed to anabiotic stress condition, such as water depravation, suboptimaltemperature, nutrient deficiency, or preferably a salt stress condition.Salt stress can be effected in many ways such as, for example, byirrigating the plants with a hyperosmotic solution, by cultivating theplants hydroponically in a hyperosmotic growth solution (e.g., Hoaglandsolution), or by culturing the plants in a hyperosmotic growth medium(e.g., MS medium). Since different plants vary considerably in theirtolerance to salinity, the salt concentration in the irrigation water,growth solution, or growth medium is preferably adjusted according tothe specific characteristics of the specific plant cultivar or variety,so as to inflict a mild or moderate effect on the physiology and/ormorphology of the plants (for guidelines as to appropriate concentrationplease see, Bernstein and Kafkafi, Root Growth Under Salinity Stress In:Plant Roots, The Hidden Half 3rd ed. Waisel Y, Eshel A and Kafkafi U.(editors) Marcel Dekker Inc., New York, 2002, and reference therein).Following exposure to the stress condition the plants are frequentlymonitored until substantial physiological and/or morphological effectsappear in wild type plants. Subsequently, transformed plants notexhibiting substantial physiological and/or morphological effects, orexhibiting higher biomass than wild-type plants, are identified asabiotic stress tolerant plants.

Although stable transformation is presently preferred, transienttransformation of leaf cells, meristematic cells or the whole plant isalso envisaged by the present invention.

Transient transformation can be effected by any of the direct DNAtransfer methods described above or by viral infection using modifiedplant viruses.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications inMolecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, NewYork, pp. 172-189 (1988). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO87/06261.

Preferably, the virus of the present invention is avirulent and thus isincapable of causing severe symptoms such as reduced growth rate,mosaic, ring spots, leaf roll, yellowing, streaking, pox formation,tumor formation and pitting. A suitable avirulent virus may be anaturally occurring avirulent virus or an artificially attenuated virus.Virus attenuation may be effected by using methods well known in the artincluding, but not limited to, sub-lethal heating, chemical treatment orby directed mutagenesis techniques such as described, for example, byKurihara and Watanabe (Molecular Plant Pathology 4:259-269, 2003),Gal-on et al. (1992), Atreya et al. (1992) and Huet et al. (1994).

Suitable virus strains can be obtained from available sources such as,for example, the American Type culture Collection (ATCC) or by isolationfrom infected plants. Isolation of viruses from infected plant tissuescan be effected by techniques well known in the art such as described,for example by Foster and Tatlor, Eds. “Plant Virology Protocols: FromVirus Isolation to Transgenic Resistance (Methods in Molecular Biology(Humana Pr), Vol 81)”, Humana Press, 1998. Briefly, tissues of aninfected plant believed to contain a high concentration of a suitablevirus, preferably young leaves and flower petals, are ground in a buffersolution (e.g., phosphate buffer solution) to produce a virus infectedsap which can be used in subsequent inoculations.

Construction of plant RNA viruses for the introduction and expression ofnon-viral exogenous polynucleotide sequences in plants is demonstratedby the above references as well as by Dawson, W. O. et al., Virology(1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French etal. Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters(1990) 269:73-76.

When the virus is a DNA virus, suitable modifications can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression inplants of non-viral exogenous polynucleotide sequences such as thoseincluded in the construct of the present invention is demonstrated bythe above references as well as in U.S. Pat. No. 5,316,931.

In one embodiment, a plant viral polynucleotide is provided in which thenative coat protein coding sequence has been deleted from a viralpolynucleotide, a non-native plant viral coat protein coding sequenceand a non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral polynucleotide, andensuring a systemic infection of the host by the recombinant plant viralpolynucleotide, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native polynucleotidesequence within it, such that a protein is produced. The recombinantplant viral polynucleotide may contain one or more additional non-nativesubgenomic promoters. Each non-native subgenomic promoter is capable oftranscribing or expressing adjacent genes or polynucleotide sequences inthe plant host and incapable of recombination with each other and withnative subgenomic promoters. Non-native (foreign) polynucleotidesequences may be inserted adjacent the native plant viral subgenomicpromoter or the native and a non-native plant viral subgenomic promotersif more than one polynucleotide sequence is included. The non-nativepolynucleotide sequences are transcribed or expressed in the host plantunder control of the subgenomic promoter to produce the desiredproducts.

In a second embodiment, a recombinant plant viral polynucleotide isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral polynucleotide isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral polynucleotide. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native polynucleotidesequences may be inserted adjacent the non-native subgenomic plant viralpromoters such that the sequences are transcribed or expressed in thehost plant under control of the subgenomic promoters to produce thedesired product.

In a fourth embodiment, a recombinant plant viral polynucleotide isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral polynucleotide to produce a recombinant plantvirus. The recombinant plant viral polynucleotide or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral polynucleotide is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(exogenous polynucleotide) in the host to produce the desired protein.

Techniques for inoculation of viruses to plants may be found in Fosterand Taylor, eds. “Plant Virology Protocols: From Virus Isolation toTransgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol81)”, Humana Press, 1998; Maramorosh and Koprowski, eds. “Methods inVirology” 7 vols, Academic Press, New York 1967-1984; Hill, S. A.“Methods in Plant Virology”, Blackwell, Oxford, 1984; Walkey, D. G. A.“Applied Plant Virology”, Wiley, New York, 1985; and Kado and Agrawa,eds. “Principles and Techniques in Plant Virology”, VanNostrand-Reinhold, New York.

In addition to the above, the polynucleotide of the present inventioncan also be introduced into a chloroplast genome thereby enablingchloroplast expression.

A technique for introducing exogenous polynucleotide sequences to thegenome of the chloroplasts is known. This technique involves thefollowing procedures. First, plant cells are chemically treated so as toreduce the number of chloroplasts per cell to about one. Then, theexogenous polynucleotide is introduced via particle bombardment into thecells with the aim of introducing at least one exogenous polynucleotidemolecule into the chloroplasts. The exogenous polynucleotides selectedsuch that it is integratable into the chloroplast's genome viahomologous recombination which is readily effected by enzymes inherentto the chloroplast. To this end, the exogenous polynucleotide includes,in addition to a gene of interest, at least one polynucleotide stretchwhich is derived from the chloroplast's genome. In addition, theexogenous polynucleotide includes a selectable marker, which serves bysequential selection procedures to ascertain that all or substantiallyall of the copies of the chloroplast genomes following such selectionwill include the exogenous polynucleotide. Further details relating tothis technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507which are incorporated herein by reference. A polypeptide can thus beproduced by the protein expression system of the chloroplast and becomeintegrated into the chloroplast's inner membrane.

Since the traits of the present invention (e.g., NUE and abiotic stresstolerance) in plants can involve multiple genes acting additively or insynergy (see, for example, in Quesda et al., Plant Physiol. 130:951-063,2002), the present invention also envisages expressing a plurality ofexogenous polynucleotides in a single host plant to thereby achievesuperior NUE, tolerance, biomass and/or yield.

Expressing a plurality of exogenous polynucleotides in a single hostplant can be effected by co-introducing multiple nucleic acidconstructs, each including a different exogenous polynucleotide, into asingle plant cell. The transformed cell can than be regenerated into amature plant using the methods described hereinabove.

Alternatively, expressing a plurality of exogenous polynucleotides in asingle host plant can be effected by co-introducing into a singleplant-cell a single nucleic-acid construct including a plurality ofdifferent exogenous polynucleotides. Such a construct can be designedwith a single promoter sequence which can transcribe a polycistronicmessage including all the different exogenous polynucleotide sequences.To enable co-translation of the different polypeptides encoded by thepolycistronic message, the polynucleotide sequences can be inter-linkedvia an internal ribosome entry site (IRES) sequence which facilitatestranslation of polynucleotide sequences positioned downstream of theIRES sequence. In this case, a transcribed polycistronic RNA moleculeencoding the different polypeptides described above will be translatedfrom both the capped 5′ end and the two internal IRES sequences of thepolycistronic RNA molecule to thereby produce in the cell all differentpolypeptides. Alternatively, the construct can include several promotersequences each linked to a different exogenous polynucleotide sequence.

The plant cell transformed with the construct including a plurality ofdifferent exogenous polynucleotides, can be regenerated into a matureplant, using the methods described hereinabove.

Alternatively, expressing a plurality of exogenous polynucleotides in asingle host plant can be effected by introducing different nucleic acidconstructs, including different exogenous polynucleotides, into aplurality of plants. The regenerated transformed plants can then becross-bred and resultant progeny selected for superior traits such asNUE, abiotic stress tolerance and/or biomass, using conventional plantbreeding techniques.

As mentioned hereinabove, transgenic plants of the present invention canbe used for improving myriad of commercially desired traits which areall interrelated as is discussed hereinbelow.

As used herein the term “trait” refers to a characteristic or quality ofa plant which may overall (either directly or indirectly) improve thecommercial value of the plant.

As used herein the term “improving” or “increasing” refers to improvingor increasing the trait of the transgenic plant of the present inventionby at least about 2% more, 5% more, 10% more, 20% more, 30% more, 40%more, 50% more, 60% more, 70% more, 80% more, 90% or more than that ofthe non-transgenic plant (e.g., mock transfected, or naïve).

The following are illustrative examples of traits which can be improvedusing the polynucleotides or polypeptides of the present invention.

Fertilizer use efficiency—Although the following will be more elaboratedregarding Nitrogen (N) Use Efficiency (NUE) it is to be understood thatthe present invention envisages increasing/improving the overallfertilizer use efficiency of all minerals and organic moieties absorbedfrom the soil, such as phosphates (PUE) and potassium (KUE) by thetransgenic plants of the present invention.

The efficiency by which N is used by the plant is affected among otherthings by both N uptake efficiency and N utilization efficiency. Theratio of the amount of N uptake by the plant (or N content, kg N) overthe amount of N supplied/applied (kg N_(s)) is an uptake efficiency (kgN kg⁻¹ N_(s)), while the grain yield (kg grain) to N uptake (kg N) ratiois N utilization efficiency (NUE, kg grain kg⁻¹ N). See Moll 1982Analysis and interpretation of factors which contribute to efficiency ofnitrogen utilization. Argon. J. 74:562-564. NUE is also affected by Nlosses through ammonia volatilization, nitrification-denitrification,leaching and runoff which decrease the availability of N to the plant.

The present inventors realized that genes which alter root architecturecan be used to improve NUE. The rationale is to position a higherfraction of the roots in deeper soil layers where the fertilizer isleached or increasing the soil coverage where the fertilizer isconcentrated. This strategy was already proven successful withPhosphorous deficiency in soybean (Miller, C R, I Ochoa, K L Nielsen, DBeck, J P Lynch. 2003. Genetic variation for adventitious rooting inresponse to low phosphorus availability: potential utility forphosphorus acquisition from stratified soils. Functional Plant Biology30:973-985) and maize (Zhu, J, JP Lynch. 2004. The contribution oflateral rooting to phosphorus acquisition efficiency in maize (Zea maysL.) seedlings. Functional Plant Biology 31:949-958) and other cropplants. Root morphogenesis is dramatically affected by developmentprogramming as well as by environmental conditions. Drought leads to apronounced downward development of the root structure to reach waterlocated in deeper soil layers (see FIG. 1A) while local nutrientavailability causes a local root outgrowth increasing the totalabsorptive surface of the root system. The development of root systemsis usually highly asymmetric and reflects the ability of roots to adjusttheir growth and development to environmental factors. Genes and geneexpression control the developmental changes, as is the case of ANR1: aputative transcription factor with a role in NO₃ ⁻ signaling. When ANR1is down-regulated by antisense or co-suppression, the resultingtransgenic lines are defective in their root response to localizedsupplies of NO₃ ⁻ (Zhang, H., Forde, B. G. 1998, An Arabidopsis MADS boxgene that controls nutrient-induced changes in root architecture,Science 270:407). Hence, altering expression of the polynucleotides ofthe present invention in transgenic plants may have desirable effects onroot morphogenesis, some of them could positively affect NUE andabiotic/biotic stress tolerance and/or to enhance plant yield and vigor.Specific examples include FUE_7, FUE_16 and FUE_34_Evo.

Additionally selected are those which enhance intracellular storagereduce the cytosolic concentration, thereby reducing the energeticbarrier for the membrane transporters responsible for the uptake of thedifferent forms of the fertilizer. In this way, higher rates offertilizer uptake can be obtained. Similarly, as expected from anyenzymatic reaction or pathway, if the product of fertilizer assimilationis efficiently removed for the cellular milieu the enzymatic pathwaybuilding this product is expected to occur at accelerated rate leadingto improved assimilation or use efficiency.

In yet another approach to improve FUE, a third group of genes wasidentified. Those genes are related to the biochemical pathways involvedin the conversion of the inorganic fertilizer form to the organicmaterial (assimilation process). Releasing bottlenecks in theassimilation process has granted promising effects on enhanced nutrientsuse efficiency, as in the case of the transcription factor Dof1(Yanagisawa Sd, Akiyama A, Kisaka H, Uchimiya H, Miwa T. Metabolicengineering with Dof1 transcription factor in plants: Improved nitrogenassimilation and growth under low-nitrogen conditions. Proc Natl AcadSci USA. 2004 May 18;101(20):7833-8). The genes found using thisapproach relieve the biochemical bottlenecks leading to enhancedfertilizer use efficiency. FUE_101 and FUE_102 comprise a transporteractivity and therefore may be used to improve nutrient uptake.

Storage capacity—polynucleotide and polypeptide sequences of the presentinvention are directed to enhance the general storage capability of theplant. The capacity for grain production is predetermined by the plantsability to absorb and store mineral nitrogen in its early phases ofdevelopment (Hirel et el. Plant Physiol, March 2001, Vol. 125, pp.1258-1270). Therefore enhanced storage capacity in the form of nitrateor amino acids highly likely will increase the grain yield. Thisinvention includes molecular mechanisms to improve the nitrogen or anymetabolic nutrient storage capacity of the plant. Examples include,amino acids, protein, starch and oil, fiber, ash, chlorophyll andmineral content.

Plant protein content is directly related to the N concentration in thegrain (see Mosse 1990 J. Agric. Food Chem. 38:18-24). This is highlyvaluable for improving the nutritional value of food. For example,children consuming high-protein (10% milled rice showed improved growthcompared with children consuming average-protein (6-7%) milled rice[Juliano (1993) Rice in Human Nutrition. IRRI and FAO, Rome]. Thus,transgenic plants displaying high efficiency in remobilizing N fromvegetative parts to the edible part (e.g., grain) are highly valuable.Since the levels of nitrogen at flowering determine the grain yield,overexpression of the polynucleotide sequences of the present inventionwill improve the grain yield and/or enhance protein content of grain atthe entire plant level. In addition, increased solute content of theplant prevents evaporation and water loss due to heat, drought,salinity, osmoticum, etc therefore providing a better plant tolerance tothe above stresses. Overexpression of FUE_501, FUE_502, FUE_503, FUE_504or FUE_49, FUE_51, FUE_52, FUE_53 or FUE_100 and FUE_101 are expected toincrease the storage capacity of the tissues where they are expressed.This may lead to a stronger sink capacity, and to a better use of thefertilizer applied (particularly nitrogen) due to enhanced absorptionfrom the soil. Since the levels of nitrogen at flowering determine thegrain yield, it is expected that the overexpression of the above geneswill also improve the grain yield and/or enhance protein content ofgrain and the entire plant protein level. In addition, increased solutecontent of the plant will prevent evaporation and water loss due toheat, drought, salinity, osmoticum, and the like thereby providing abetter plant tolerance to the above stresses.

Delayed leaf senescence or “stay-green”—extends nutrient uptake longerinto the plant cycle providing increased capacity to store nitrogen andto provide photosynthate during grain filling. If a plant has more greenleaves at the period of pollination more nitrogen will be available forredistribution to the kernels causing an improvement in fertilizer useefficiency and probably grain yield. Some of the genes presented in thisinvention increase the “stay green” trait or exhibit enhanced growth(faster growth and/or bigger leaves and stalks) as a mechanism toimprove fertilizer use efficiency, grain yield and overall stresstolerance of the plant.

Invertase is an enzyme that is known to delay senescence when expressedunder a senescence-activated promoter (Balibrea Lara, et al. Plant Cell16: 1276-1287, 2004). FUE_30 and FUE_31 have a predicted invertaseactivity and therefore can extend the stay-green characteristics of theplant and therefore increase the overall capacity to store nitrogen inplants for redistribution during grain filling.

Cytokinin is involved, among other processes, in the inhibition of leafsenescence. FUE_55 is a gene with a striking similarity to tRNAisopentenyltransferase, an important enzyme in the cytokinin metabolicpathway. Increased expression of FUE_55 in the roots or shoots willincrease the levels of cytokinin leading to enhanced leaf expansion andcell duplication, delayed senescence, increased sink strength of thetissues, enhanced nitrogen utilization, etc.

Zeatin is a naturally occurring cytokinin. FUE_505 is a putative AP2domain-containing transcription factor that based on microarrayexperiments it clusters tightly together with ARR genes (Arabidopsisresponse regulators) which mediate the response of the shoot tocytokinin. ARR genes are Zeatin responsive and are induced afternitrogen addition to nitrogen starved plants. FUE_505 co-regulationtogether with ARRs indicates a role in the cytokinin response. It ishighly likely that constitutive expression of FUE_505 will delay leafsenescence and improve nitrogen utilization and plant growth due tocontinuous activation of the cytokinin response of the shoot.

Stress tolerance—Transgenic plants of the present invention are expectedto exhibit tolerance to biotic and abiotic stress.

The phrase “stress” used herein refers to any adverse effect onmetabolism, growth, reproduction and/or viability of a plant.Accordingly, abiotic stress can be induced by suboptimal environmentalgrowth conditions such as, for example, salinity, water deprivation,flooding, freezing, low or high temperature, heavy metal toxicity,anaerobiosis, nutrient deficiency (including also nutrientinaccessibility such as due to leaching), atmospheric pollution or UVirradiation. Biotic stress can be induced for example by pathogens whichare found in the environment.

The phrase “stress tolerance” as used herein refers to the ability of aplant to endure an stress (abiotic) without suffering a substantialalteration in metabolism, growth, productivity and/or viability.Preferably, the genetically engineered plants of the present inventionexhibit at least about 2% more, 5% more, 10% more, 20% more, 30% more,40% more, 50% more, 60% more, 70% more, 80% more, 90% more or evenhigher tolerance to abiotic stress than non-transgenic plants.

Plants are subject to a range of environmental challenges. Several ofthese, including salt stress, general osmotic stress, drought stress andfreezing stress, have the ability to impact whole plant and cellularwater availability. Not surprisingly, then, plant responses to thiscollection of stresses are related. In a recent review, Zhu notes that“most studies on water stress signaling have focused on salt stressprimarily because plant responses to salt and drought are closelyrelated and the mechanisms overlap” (Zhu (2002) Ann. Rev. Plant Biol.53: 247-273). Many examples of similar responses and pathways to thisset of stresses have been documented. For example, the CBF transcriptionfactors have been shown to condition resistance to salt, freezing anddrought (Kasuga et al. (1999) Nature Biotech. 17: 287-291). TheArabidopsis rd29B gene is induced in response to both salt anddehydration stress, a process that is mediated largely through an ABAsignal transduction process (Uno et al. (2000) Proc. Natl. Acad. Sci.USA 97: 11632-11637), resulting in altered activity of transcriptionfactors that bind to an upstream element within the rd29B promoter. InMesembryanthemum crystallinum (ice plant), Patharker and Cushman haveshown that a calcium-dependent protein kinase (McCDPK1) is induced byexposure to both drought and salt stresses (Patharker and Cushman (2000)Plant J. 24: 679-691). The stress-induced kinase was also shown tophosphorylate a transcription factor, presumably altering its activity,although transcript levels of the target transcription factor are notaltered in response to salt or drought stress. Similarly, Saijo et al.demonstrated that a rice salt/drought-induced calmodulin-dependentprotein kinase (OsCDPK7) conferred increased salt and drought toleranceto rice when overexpressed (Saijo et al. (2000) Plant J. 23: 319-327).

Exposure to dehydration invokes similar survival strategies in plants asdoes freezing stress (see, for example, Yelenosky (1989) Plant Physiol89: 444-451) and drought stress induces freezing tolerance (see, forexample, Siminovitch et al. (1982) Plant Physiol 69: 250-255; and Guy etal. (1992) Planta 188: 265-270). In addition to the induction ofcold-acclimation proteins, strategies that allow plants to survive inlow water conditions may include, for example, reduced surface area, orsurface oil or wax production. In another example increased solutecontent of the plant prevents evaporation and water loss due to heat,drought, salinity, osmoticum, and the like therefore providing a betterplant tolerance to the above stresses.

It will be appreciated that some pathways involved in resistance to onestress (as described above), will also be involved in resistance toother stresses, regulated by the same or homologous genes. Of course,the overall resistance pathways are related, not identical, andtherefore not all genes controlling resistance to one stress willcontrol resistance to the other stresses. Nonetheless, if a geneconditions resistance to one of these stresses, it would be apparent toone skilled in the art to test for resistance to these related stresses.Methods of assessing stress resistance are further provided in theExamples section which follows.

The ability of the transgenic plants of the present invention to endurestress is expected to affect plant biomass, vigor and yield. Theopposite is also anticipated to present good results, essentially,improved biomass, vigor and/or yield is expected to improve theendurance of transgenic plants of the present invention to stressconditions.

As used herein the phrase “plant biomass” refers to the amount orquantity of tissue produced from the plant in a growing season, whichcould also determine or affect the plant yield or the yield per growingarea.

As used herein the phrase “plant vigor” refers to the amount or quantityof tissue produced from the plant in a given time. Hence increase vigorcould determine or affect the plant yield or the yield per growing timeor growing area.

As used herein the phrase “plant yield” refers to the amount or quantityof tissue produced and harvested as the plant produced product. Henceincrease yield could affect the economic benefit one can obtain from theplant in a certain growing time.

To analyze the effect of the transgene on plant physiology, one canassess overall yield and biomass, the tolerance of the plants tofertilizer deficiency and to abiotic stresses such as drought, salinity,cold and heat stresses, freezing, etc. Also of great importance is toassess whether the plant at any of its parts contains an increasedcontent of protein, free amino acids, oil and any other metaboliccompounds of value.

The following summarizes assays which may be used to qualify transgenicplants or transgenes of the present invention (further description ofthese and other assays are provided in the Examples section whichfollows).

Fertilizer use efficiency—To analyze whether the transgenic plants aremore responsive to fertilizers, plants are grown in agar plates or potswith a limited amount of fertilizer. The plants are analyzed for theiroverall size, time to flowering, yield, protein content of shoot and/orgrain. An example methodology of a test for fertilizer use efficiency isprovided in the work by Yanagisawa et al (Proc Natl Acad Sci USA. 2004;101:7833-8) in which seeds of transgenic Arabidopsis are checked forgrowth rates under limiting-nitrogen conditions (for example at 0.01 mM,0.05 mM 0.15 mM, 0.3 mM, 1 mM, 3 mM Nitrogen in the form of nitrate orammonium). The parameters checked are the overall size of the matureplant, its wet and dry weight, the weight of the seeds yielded, theaverage seed size and the number of seeds produced per plant. Otherparameters that may be tested are: the chlorophyll content of leaves (asnitrogen plant status and the degree of leaf verdure is highlycorrelated), amino acid and the total protein content of the seeds orother plant parts such as leaves or shoots, oil content, etc. Similarly,instead of providing nitrogen at limiting amounts, Phosphate orPotassium can be added at increasing concentrations. Again, the sameparameters measured are the same as listed above. In this way, besidesNitrogen Use Efficiency (NUE), Phosphate Use Efficiency (PUE) andPotassium Use Efficiency (KUE) are assessed checking the ability of thetransgenic plants to thrive under nutrient restraining conditions.

Nitrogen determination—The procedure for N concentration determinationin the structural parts of the plants involves the potassium persulfatedigestion method to convert organic N to NO₃ ⁻ (Purcell and King 1996Argon. J. 88:111-113, the modified Cd⁻ mediated reduction of NO₃ ⁻ toNO₂ ⁻ (Vodovotz 1996 Biotechniques 20:390-394) and the measurement ofnitrite by the Griess assay (Vodovotz 1996, supra). The absorbancevalues are measured at 550 nm against a standard curve of NaNO₂. Theprocedure is described in details in Samonte et al. 2006 Agron. J.98:168-176.

Grain protein concentration—Grain protein content (g grain protein m⁻²)is estimated as the product of the mass of grain N (g grain N m⁻²)multiplied by the N/protein conversion ratio of k-5.13 (Mosse 1990,supra). The grain protein concentration is estimated as the ratio ofgrain protein content per unit mass of the grain (g grain protein kg⁻¹grain).

Oil content—The amount of oil expressed as percentage of dry weight. Oilcontent is defined as the maximum amount of material (lipid) that can beremoved from the seed by extraction with specific solvents (usuallyhexane or petroleum ether). Oil content is measured directly by grindingthe seed and extracting the oil in a continuous extractor. Indirect oilcontent analysis can be carried out using either Nuclear MagneticResonance (NMR) Spectroscopy or Near Infrared (NI) Spectroscopy. The NMRtechnique measures the resonance energy absorbed by hydrogen atoms inthe liquid state of the sample while NI utilizes the absorption of nearinfrared energy (1100-2500 nm) by the sample. While the precision of NIRmethods is not as good as extraction methods, NMR methods give veryaccurate and precise results when calibrated carefully.

Drought tolerance assay/Osmoticum assay—Tolerance to drought isperformed to identify the genes conferring better plant survival afteracute water deprivation. To analyze whether the transgenic plants aremore tolerant to drought, an osmotic stress produced by the non-ionicosmolyte sorbitol in the medium can be performed. Control and transgenicplants are germinated and grown in plant-agar plates for 4 days, afterwhich they are transferred to plates containing 500 mM sorbitol. Thetreatment causes growth retardation, then both control and transgenicplants are compared, by measuring plant weight (wet and dry), yield, andby growth rates measured as time to flowering.

Conversely, soil-based drought screens are performed with plantsoverexpressing the candidate genes detailed above. Seeds from controlArabidopsis plants, or other transgenic plants overexpressing orsilencing a polypeptide of the invention are germinated and transferredto pots. Drought stress is obtained after irrigation is ceasedaccompanied by placing the pots on absorbent paper to enhance thesoil-drying rate. Transgenic and control plants are compared to eachother when the majority of the control plants develop severe wilting.Plants were re-watered after obtaining a significant fraction of thecontrol plants displaying a severe wilting. Plants are ranked comparingto controls for each of two criteria: tolerance to the droughtconditions and recovery (survival) following re-watering.

Salinity tolerance assay—transgenic plants with tolerance to high saltare expected to exhibit better germination, seedling vigor or growth inhigh salt. Plants differ in their tolerance to NaCl depending on theirstage of development, therefore seed germination, seedling vigor, andplant growth responses are evaluated. A salinity tolerance test istaking plants at different developmental stages and irrigate them withincreasing concentrations of NaCl (for example 50 mM, 100 mM, 200 mM,400 mM) applied from the bottom and from above to ensure even dispersalof salt. Transgenic plants are compared to control plants in theirexternal phenotypic appearance, degree of wilting, and overall successto reach maturity and yield progeny at concentrations inhibitory tocontrol plants. Quantitative parameters of tolerance measured are, theaverage wet and dry weight, and the weight of the seeds yielded, theaverage seed size and the number of seeds produced per plant. Osmoticstress assays (including NaCl and mannitol assays) are conducted todetermine if an osmotic stress phenotype was NaCl-specific or if it wasa general osmotic stress related phenotype. Plants tolerant to osmoticstress could also have more tolerance to drought and/or freezing. Forsalt and osmotic stress germination experiments, the medium issupplemented for example with 50 mM, 100 mM, 200 mM NaCl or 100 mM, 200mM NaCl, 400 mM mannitol.

Cold stress tolerance—To analyze cold stress, mature (25 day old) plantsare transferred to 4° C. chambers for 1 or 2 weeks, with constitutivelight. Later on plants are moved back to greenhouse. Two weeks laterdamages from chilling period, resulting in growth retardation and otherphenotypes, are compared between both control and transgenic plants, bymeasuring plant weight (wet and dry), and by comparing growth ratesmeasured as time to flowering, plant size, yield, etc.

Heat stress tolerance—heat stress tolerance is achieved by exposing theplants to temperatures above 34° C. for a certain period. Planttolerance is examined after transferring the plants back to 22° C. forrecovery and evaluation after 5 days relative to internal controls(non-transgenic plants) or plants not exposed to neither cold or heatstress.

Germination tests compare the percentage of seeds from transgenic plantsthat could complete the germination process to the percentage of seedsfrom control plants that are treated in the same manner. Normalconditions are considered for example, incubations at 22° C. under22-hour light 2-hour dark daily cycles. Evaluation of germination andseedling vigor is conducted between 4 and 14 days after planting. Thebasal media is 50% Murashige-Skoog medium (MS)+vitamins.

Germination is checked also at unfavorable conditions such as cold(incubating at temperatures lower than 10° C. instead of 22° C.) orusing seed imbibition solutions that contain high concentrations of anosmolyte such as sorbitol (at concentrations of 50 mM, 100 mM, 200 mM,300 mM, 500 mM, and up to 1000 mM) or applying increasing concentrationsof salt (of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM NaCl).

Methods of assaying plant vigor, yield and biomass are well known in theart (see Example 9 and Example 10).

The present invention is also valuable for breeding and ornamentalpurposes. It is thus envisaged that polynucleotides and polypeptidesequences of the present invention which are associated with rootarchitecture (FUE_34_Evo, SEQ ID NO: 54, see also Example 11 and FIG. 8)may also be used to govern plant tillering and as such may beupregulated or downregulated to govern branching and tillering. Methodsof downregulating gene expression in plants are well known in the art.

Thus, the present invention is of high agricultural value for promotingcommercially desired traits in crop-plants.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Polynucleotide Identification and Gene Role Prediction UsingBioinformatics and Molecular Tools

Polynucleotides, suitable for increasing FUE and/or abiotic or bioticstress tolerance were identified by in-depth analysis of RNA expressionprofiles, sequence similarities, gene annotations, biochemical pathways,DNA, ESTs, protein and expression databases, which are publiclyavailable and used as input data to a series of proprietarycomputational algorithms that allow expert data mining.

Trait association was performed using accurate expression profiling bymeans of quantitative Real Time PCR, to correlate between the expressionlevels in different tissues and under specific growth conditions forelucidating the function of the expressed polynucleotide.

Materials and Methods

Identifying Polynucleotide Sequences Associated with Increased FUE

The present methodology is based on three stages described in thefollowing examples, essentially the bioinformatic filtering, molecularanalysis and in planta validation. Infra is a description of thebioinformatic algorithm.

A. Bioinformatic Filtering:

Clustering DNA sequences into gene clusters—The aim of EST (ExpressedSequence Tag) clustering is to incorporate all ESTs that share atranscript or gene parent to the same cluster. Typically, clustered ESTsare assembled into one or more consensus sequences (contigs) thatreflect the transcript diversity, providing these contigs such that theinformation they contain most truly reflects the sampled biology. A genecluster is fragmented, EST data and (if known) gene mRNA sequence data,consolidated, placed in correct context and indexed by gene such thatall expressed data concerning a single gene is in a single index class,and each index class contains the information for only one gene (Burkeet al, 1999, D2_cluster: A Validated Method for Clustering EST andFull-length cDNA Sequences. Genome Research, 9(11), 1135-1142). TheCompugen LEADS™ platform (Hypertext Transfer Protocol://World Wide Web(dot) cgen (dot) com/research/Publications/LEADSwhitepaper.pdf) was usedfor the assembly.

Calculating the digital expression for all the gene clusters in severalplant species—Digital expression, also known as electronic northernblot, compares occurrences of large number of random ESTs fromnon-normalized cDNA libraries. The variation in the relative frequencyof those tags, stored in databases, is then used to elucidate thedifferential expression of the corresponding genes. Digital Northerndata can be used to provide quantitative assessment of differentialexpression between different organs in the plant or at differentphysiological states (stress versus normal). This tool displays virtualexpression profiles of genes based on the EST sequences forming thecluster. The tool can provide the expression profile of a cluster interms of plant anatomy (i.e., in what tissues/organs is the geneexpressed), developmental stage (i.e., the developmental stages at whicha gene can be found) and profile of treatment (i.e., provides thephysiological conditions under which a gene is expressed such asdrought, cold, pathogen infection and the like). Given a randomdistribution of ESTs in the different clusters, the digital expressionprovides a probability value that describes the probability of a clusterhaving a total of N ESTs to contain X ESTs from a certain collection oflibraries. For the probability calculations the following is taken intoconsideration: a) the number of ESTs in the cluster, b) the number ofESTs originating from a specific library or a group of relatedlibraries, c) the overall number of ESTs available representing thespecies. In this way clusters with low probability values are highlyenriched with ESTs from the group of libraries of interest indicating aspecialized expression. In addition, a proprietary vocabulary (limitedcompendium of terms) is used that takes into consideration theannotation of each EST library and uses specific keywords to describethe experimental data associated with the sequences with regards totissue type, treatment and developmental stages. The terms chosen fromthe proprietary vocabulary are combined with the calculated digitalexpression to build an expression profile for each specific gene, basedon the source of the libraries that provide the sequences to the genecluster. A statistic and graphic representation of this profile is builtfor the digital expression calculations. Because the annotations arefrom a controlled vocabulary, the entire database is dissected withspecific keywords describing specific tissues, developmental stages ortreatments.

Integrating all relevant data for each gene, including expression datafrom microarray experiments, digital expression, annotation, ontology,gene families, conserved motifs and the like—A proprietary tool thatcreates groups of orthologous genes from genomic databases from multipleplant species is used. In order to provide further support to the genesidentified, orthologous genes or the orthologous group is tested as awhole to see involvement of the trait of interest according to thekeywords that are most significant in their calculated digitalexpression.

Gene selection—This is done according to several biologically soundassumptions to query the database described above. Gene selection isdone by filtering specific groups of genes using carefully selectedcriteria. The final set of genes should contain a limited number ofgenes, which one could handle in the following molecular analysis andtransgenic experiments. The different criteria utilized herein aredescribed with great detail for each gene below.

Gene selection for this invention takes into consideration severalconcepts widely accepted in plant science. Nutrient deficiencies causeadaptations of the root architecture, particularly notably for exampleis the root proliferation within nutrient rich patches to increasenutrient uptake. Nutrient deficiencies cause also the activation ofplant metabolic pathways which maximize the absorption and assimilationprocesses. In this process, genes are triggered and activated causingthe architectural change (Jose Lopez-Bucio et al, The role of nutrientavailability in regulating root architecture, Current Opinion in PlantBiology 2003, 6:280-287). Engineering the expression of the triggeredgenes may cause the plant to exhibit the architectural changes andenhanced metabolism also under other conditions. Second, it is widelyknown that the plants usually respond to drought by creating a deeperroot system that allows access to moisture located in deeper soil layers(Morgan, J. M., and A. G. Condon. 1986. Water use, grain yield, andosmoregulation in wheat. Aust. J. Plant Physiol. 13:523-532 and YiweiJiang and Bingru Huang, 2001, Crop Science 41:1168-1173). Triggeringthis effect will allow the plants to access nutrients located in deepersoil horizons particularly those readily dissolved in water likenitrates. Third, different abiotic stresses (e.g., drought, osmoticum,cold, heat, radiation, salinity, nutrient deficiencies, and the like)elicit their response using both: stress-specific as well as commonstress pathways (Gabriela M. Pastori and Christine H. Foyer, CommonComponents, Networks, and Pathways of Cross-Tolerance to Stress, PlantPhysiol, June 2002, Vol. 129, pp. 460-468). This provides thepossibility to distinguish between the genes that are involved in eachpathway.

The plant response to stress is costly in terms of energy as suchaffecting plant yield. Precise engineering of specific genes providesthe ability to activate only partially a stress response without causingthe concomitant loss in yield. Accordingly, several queries todistinguish the genes that are activated during the common andstress-specific responses were performed. Since some of the plantresponses such as enhanced root system or superior storage capabilitiesare highly preferred also at optimal growing conditions, the presentinvention envisages to create modified plants with improved FUE thatshow in addition also enhanced response to other abiotic plant stressesthat cause adverse effects on yield.

For the purpose of the identification FUE improving genes, 416899 ESTand 23563 mRNA transcript sequences from Maize were extracted fromGenbank release 145 (December 2004), cleaned by screening for vectors,low-complexity sequences and known repeats from public databases, andthen clustered and assembled into contigs using Compugen's LEADSplatform.

The EST libraries in Genbank 146 for Maize and other species such asSoybean, Tomato, Barley, Sorghum, Rice, Cotton, Wheat and Arabidopsiswere examined and annotated using keywords describing the plant tissueused, the treatment applied to grow the plants and the developmentalstage of the plants when the tissues were taken. A significance valuewas assigned to the frequency of ESTs in each contig from each ESTlibrary or set of libraries sharing the same annotation. Thesignificance value was based on a statistical test that takes intoaccount the number of ESTs from the given category in the contig, thenumber of these ESTs in the whole production, the size of the contig andthe total number of ESTs in the production.

In addition Arabidopsis microarray experiments, freely available at theNottingham Arabidopsis Stock Centre (NASC, Hypertext TransferProtocol://affymetrix (dot) arabidopsis (dot) info/)comprisingexperiments describing anatomy, development and various stressexperiments were integrated. To link the contigs of the LEADS Maizeproduction to the corresponding cluster from other species orthologuefinder algorithm was employed that uses among other filters, reciprocalBLAST analysis to identify the most similar gene in other plant species.

The following describes the parameters which were used for selectingeach gene. Table 1 below associates internal identifiers (NUE_XXX orFUE_XXX which are interchangeably used) to SEQ ID Nos.

TABLE 1 Internal SEQ ID NO: SEQ ID NO: Identifier (polynucleotide)(polypeptide) Plant FUE 1 1 110 maize 2 111 rice 3 112 soybean FUE 241318 113 maize FUE 3 5 114 maize 6 115 barley 7 116 rice FUE 4 8 117maize FUE 5 9 118 maize 10 119 rice FUE 40 11 120 maize (6/40) 12 121sorghum FUE 7 131323 122 maize 14 123 sorghum 15 124 barley FUE 8 161324125 maize 17 126 sorghum 18 127 rice FUE 9 191325 128 maize FUE 10 20129 maize 21 130 soybean 1 22 131 soybean 2 FUE 11 23 132 maize FUE 12241327 133 maize 25 134 barley 26 135 sorghum FUE 13 27 136 maize 28 137barley 29 138 rice FUE 14 301329 139 maize 31 140 barley FUE 15 32 141maize 33 142 barley 34 143 rice 35 144 tomato 36 145 soybean 1 FUE 16 37146 maize 38 147 barley 39 148 rice 40 149 soybean 41 150 tomato FUE 1742 151 maize 43 152 barley 44 153 sorghum 45 154 rice 46 155 soybean 147 156 soybean 2 48 157 tomato FUE_30 49 158 maize FUE_31 50 159 maizeFUE_32 51389 160 maize 52 161 wheat FUE_33 53 162 maize FUE_34_evo 54163 maize FUE_34_Pat 55 164 maize FUE_35 56 165 maize FUE_36 57 166maize FUE_37 58 167 maize 59 168 barley 60 169 wheat 61 170 sugarcane 62171 rice 63 172 soybean FUE_38 64 173 maize 65 174 barley 66 175 sorghum67 176 soybean FUE_39 68 177 maize FUE_41 69 178 maize FUE_43 70 1791186maize 71 180 rice 72 181 sorghum FUE_44 73 182 maize 74 183 soybean 75184 barley 76 185 sorghum FUE_45 77 186 maize FUE_46 78673 1871222 maizeFUE_47 79 188 maize FUE_48 80 189 maize 81 190 rice 82 191 barley 83 192sorghum FUE_49 841335 1931243? maize 85 194 sorghum FUE_50 86699 1951248maize FUE_51 87 196 maize 88 197 sorghum 89 198 barley 90 199 barley 291 200 wheat 1 92 201 wheat 2 FUE_52 93 202 maize FUE_53 94 203 maize 95204 sorghum 96 205 rice 97 206 wheat FUE_54 98 207 maize 99 208 sorghum100 209 rice FUE_55 101739 2101288 maize FUE_100 102 211 maize FUE_101103 212 maize FUE_102 104 213 maize FUE_501 105 214 Arabidopsis thalianaFUE_502 106 215 Arabidopsis thaliana FUE_503 107 216 Arabidopsisthaliana FUE_504 108 217 Arabidopsis thaliana FUE_505 109 218Arabidopsis thaliana

Of note, overexpression Fold (“Fold”) is calculated as the ratio betweenthe number of ESTs found in a gene or an orthologue group for a certaincategory (“Keyword”) and the number of expected ESTs according to anormal distribution. A probabilistic value (P-value) was estimated forthe calculated overexpression folds. Genes were selected based on theresults presented here and other computational filtering combined withmanual curation as detailed above.

FUE_1 was selected for its strong root expression in maize (as shown inTable 2 and FIGS. 2A and 2B), as well as the expression pattern of itsorthologues such as in soybean and rice (as shown in Table 3 and FIGS.2C and 2D). In addition, FUE_1 displays some homology to RCc3 protein(GENEBANK Accession NO. L27208) a known root specific protein putativelyassociated with lipid transport.

TABLE 2 Digital expression of FUE_1 in maize: Anatomy Primary SecondaryESTs ESTs Key- Key- in in Pro- Expected word word Gene duction ESTs Foldp-value flower 4 89278 10.8145 0.369873 0.997498 seedling + 4 90121.09165 3.66417 0.0234977 female flower leaf 1 35689 4.32312 0.2313140.989277 ear leaf 1 7960 1 1 0.622546 mix 2 90046 10.9076 0.1833590.999938 root 37 36059 4.36794 8.47082 1.78E−15 primary 24 33886 4.104725.84693 7.82E−14 root system root tip 13 2173 1 13 2.22E−15 seedling 632466 3.93271 1.52567 0.19702 seedling + 4 9012 1.09165 3.664170.0234977 female flower shoot 1 16152 1.95654 0.511106 0.864469

TABLE 3 Digital expression of FUE_1 ortholog group: Anatomy cellgerminating callus cambium suspension flower seed leaf mix phloem podroot seedling cotton 0.26 grape 0.39 maize 0.07 0.06 0.4 2.61 0.2 poplar2.8 4.1 rice 0.9 0.08 5.25 1.24 sorghum 0.46 2 0.21 soybean 1 0.31 0.11tomato 1.46 TOTAL 1.92 2.8 2 0.47 0.9 0.14 0.4 4.1 1 8.17 2.02

FUE_2 is a protein of unknown function displaying a digital expressionlinking it to roots and particularly to roots under drought stress.

FUE_3 is a putative non-specific lipid transfer protein expressedparticularly in roots in maize (Table 4 and Table 5), but also in barleyand rice. Its expression in roots under drought is conserved throughoutevolution in sorghum, maize, rice and barley (as shown in Table 6 and7). There are indications that the gene is expressed also under salinitystress in barley and rice and under nitrogen deficiency in barley.

TABLE 4 Digital expression of FUE_3 in maize: Anatomy Secondary ESTs inESTs in Keyword Keyword Gene Production Expected ESTs Fold p-valueflower 1 89278 7.82327 0.127824 0.999862 seedling + 1 9012 1 1 0.550246female flower mix 2 90046 7.89057 0.253467 0.998578 root 30 360593.15978 9.49432 2.66E−15 primary root 30 33886 2.96937 10.1032 0 systemseedling 2 32466 2.84494 0.703003 0.789648 seedling + 1 9012 1 10.550246 female flower shoot 1 16152 1.41537 0.706529 0.764428

TABLE 5 Digital expression of FUE_3 in maize: Treatment Secondary ESTsin ESTs in Keyword Keyword Gene Production Expected ESTs Fold p-valuedrought 30 21216 1.85912 16.1367 3.44E−15 water stress 17 6113 1 17 0 48h water stress 6 6417 1 6 1.84E−05 5 h water stress 7 2720 1 7 3.76E−095 h and 48 h, Subtracted library mix 2 36475 3.19624 0.625736 0.8422

TABLE 6 Digital expression of FUE_3 ortholog group: Anatomy germinatingculm flower seed grain leaf mix rachis root seedling shoot barley 1 0.390.19 0.03 2.24 1.4 0.18 maize 0.07 0.34 2.66 0.08 rice 0.24 0.9 0.31 0.2sorghum 0.67 TOTAL 1 0.07 0.39 0.19 0.95 0.34 2.24 4.95 0.58 0.2

TABLE 7 Digital expression of FUE_3 ortholog group: Treatment lightnutrient photoperiod drought response mix deficiencies pathogen responsesalinity waterlogged barley 0.81 0.92 0.53 0.07 1 maize 6.51 0.83 rice2.29 8 3.48 sorghum 1.61 0.34 TOTAL 11.22 0.92 0.83 0.53 0.42 8 3.48 1

FUE_4 is also a protein of unknown function displaying specific digitalexpression under drought stress (as shown in Table 8 and 9).

TABLE 8 Digital expression of FUE_4 in maize: Anatomy Key- SecondaryESTs in ESTs in Expected word Keyword Gene Production ESTs Fold p-valueroot 6 36059 1 6 6.44E−07 primary root 6 33886 1 6 4.44E−07 system

TABLE 9 Digital expression of FUE_4 in maize: Treatment ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 6 21216 1 62.67E−08 water 3 6113 1 3 7.54E−05 stress 48 h water 3 6417 1 3 8.71E−05stress 5 h

FUE_5 is a Ubiquinol-cytochrome C reductase complex like proteinexpressed under several stresses and particularly under drought andsalinity in maize (as shown in Table 10) and rice roots.

TABLE 10 Digital expression of FUE_5 in maize: Treatment Secondary ESTsin ESTs in Expected Keyword Keyword Gene Production ESTs Fold p-valuedrought 16 21216 2.67932 5.97167 3.75E−09 CONTROL 1 5966 1 1 0.532021well watered 0 h water stress 9 6113 1 9 6.92E−08 48 h water stress 46417 1 4 0.00876739 5 h water stress 2 2720 1 2 0.0465022 5 h and 48 h,Subtracted library mix 3 36475 4.60634 0.651276 0.851418 pathogen 1 22601 1 0.248934 Fusarium, 6 h 1 667 1 1 0.0808552 post infection salinity 33579 1 3 0.0105368 150 mM 3 3579 1 3 0.0105368 NaCl 24 h

FUE_6 displays homology to a multiple stress-associated zinc-fingerprotein, expressed in maize during grain development and in roots,particularly under drought stress (as shown in Tables 11 and 12respectively). The present inventors uncovered that FUE_6 and FUE_40 arepart of the same transcript. FUE_6 represents the 5′ region of thetranscript, while FUE_40 the 3′ region. For convenience, this transcriptis presented in this herein under the name FUE_40.

TABLE 11 Digital expression of FUE_6 in maize: Anatomy ESTs Key-Secondary in ESTs in Expected word Keyword Gene Production ESTs Foldp-value grain 1 73038 2.07065 0.48294 0.899148 glume 1 2043 1 10.0564193 root 10 36059 1.02228 9.78202 4.83E−10 primary 10 33886 1 102.61E−10 root system

TABLE 12 Digital expression of FUE_6 in maize: Treatment ESTs Key-Secondary in ESTs in Expected word Keyword Gene Production ESTs Foldp-value drought 10 21216 1 10 2.50E−12 CONTROL 2 5966 1 2 0.011856 wellwatered 0 h water stress 1 6113 1 1 0.16028 48 h water stress 4 6417 1 42.25E−05 5 h water stress 3 2720 1 3 5.44E−05 5 h and 48 h, Subtractedlibrary

FUE_7 is a hypothetical protein displaying similarity to antifreezeprotein expressed predominantly in barley and maize in roots and with amore evident expression during drought stress (as shown in Table 13 and14).

TABLE 13 Digital expression of FUE_7 ortholog group: Anatomy flowergrain leaf mix root seedling barley 0.96 0.33 1.86 0.21 maize 0.15 0.830.39 4.28 0.34 sorghum 1.13 TOTAL 0.15 1.79 1.46 0.39 6.14 0.55

TABLE 14 Digital expression of FUE_7 ortholog group: Treatment droughtpathogen salinity waterlogged barley 3.47 0.38 1 maize 9.64 1 sorghum7.75 TOTAL 20.87 0.38 1 1

FUE_8 displays homology to Acyl-ACP thioesterase and expressed in maizeroots under drought stress (as shown in Table 15). The sorghum and riceorthologues show also a clear association to drought as revealed by thesource of the sequences composing the genes.

TABLE 15 Digital expression of FUE_8 in maize: Anatomy ESTs Secondary inESTs in Expected Keyword Keyword Gene Production ESTs Fold p-value root6 36059 1 6 6.44E−07 primary 6 33886 1 6 4.44E−07 root system

FUE_9 is a hypothetical protein expressed in maize roots under droughtstress.

FUE_10 displays homology to electron transport-associated proteins.displaying a strong root expression solely associated to drought inmaize (as shown in Table 16 and 17) and to other abiotic stresses asfound for its orthologue expression in soybean.

TABLE 16 Digital expression of FUE_10 in maize: Anatomy Secondary ESTsin ESTs in Keyword Keyword Gene Production Expected ESTs Fold p-valueflower 1 89278 3.22135 0.310429 0.974291 seedling + 1 9012 1 1 0.280367female flower grain 1 73038 2.63537 0.379453 0.946054 pericarp 1 9389 11 0.290325 mix 2 90046 3.24906 0.615563 0.870275 root 8 36059 1.301096.1487 9.97E−06 primary root 8 33886 1.22268 6.543 6.26E−06 systemseedling 2 32466 1.17145 1.70729 0.329608 seedling + 1 9012 1 1 0.280367female flower shoot 1 16152 1 1 0.448596

TABLE 17 Digital expression of FUE_10 in maize: Treatment ESTs Key-Secondary in ESTs in Expected word Keyword Gene Production ESTs Foldp-value drought 8 21216 1 8 1.78E−07 water 5 6113 1 5 1.72E−06 stress 48h water 2 6417 1 2 0.0218099 stress 5 h water 1 2720 1 1 0.093796 stress5 h and 48 h, Subtracted library mix 2 36475 1.3161 1.51964 0.384279

FUE_11 displays homology to the physical impedance induced protein andis expressed in maize in roots also during drought stress.

FUE_12 is an unknown protein expressed in maize roots under water stress(drought) and in other abiotic stresses such as drought in barley andunder nitrogen deficiency and heat stress in sorghum.

FUE_13 displays homology to Pathogenesis-related protein 10, and isexpressed in different stresses in maize (drought, pathogens asFusarium). The barley and rice orthologues are strongly expressedparticularly under abiotic and biotic stresses linking this protein tothe ubiquitous plant response to stress.

FUE_14 is a maize protein displaying homology to Germin-like protein 6.The digital expression of its barley orthologue links this protein topathogen response, as well as drought and nitrogen deficiency response.

FUE_15 is a putative auxin-regulated protein also expressed in maizeroots. Its orthologues from barley, rice and are related to drought aswell as other biotic and abiotic stresses such as pathogen response. Intomato and soybean, the protein is linked to the response to pathogensand nutrient deficiencies.

FUE_16 is an Uclacyanin 3-like protein, expressed strongly in maizeroots under water stress (as shown in Table 18 and 19). Its orthologuesfrom barley, rice, soybean and tomato are also expressed in roots butinvolved in the response to other several biotic and abiotic stressesparticularly to light response (as shown in Table 20 and 21).

TABLE 18 Digital expression of FUE_16 in maize: Anatomy Secondary ESTsin ESTs in Keyword Keyword Gene Production Expected ESTs Fold p-valueflower 3 89278 3.91163 0.766943 0.78613 seedling + 3 9012 1 3 0.00667346female flower root 14 36059 1.57989 8.86136 1.86E−12 primary root 1433886 1.48468 9.42961 7.90E−13 system seedling 3 32466 1.42247 2.109010.165456 seedling + 3 9012 1 3 0.00667346 female flower

TABLE 19 Digital expression of FUE_16 in maize: Treatment ESTs ESTs inKey- Secondary in Pro- Expected word Keyword Gene duction ESTs Foldp-value drought 14 21216 1 14 3.77E−15 CONTROL 2 5966 1 2 0.0275854 wellwatered 0 h water stress 4 6113 1 4 0.000124308 48 h water stress 6 64171 6 2.16E−07 5 h water stress 2 2720 1 2 0.00622988 5 h and 48 h,Subtracted library

TABLE 20 Digital expression of FUE_16 ortholog group: Anatomy seed- culmflower leaf mix root seed ling shoot xylem barley 1 0.45 0.1  0.48 maize0.17 2.14 0.48 poplar 0.24 1.57 rice 0.33 3.62 0.51 sorghum 0.4  soybean0.49 0.04 0.07 0.43 3 0.71 0.46 tomato 0.48 TOTAL 1 1.11 0.14 0.41 7.413 2.11 0.46 1.57

TABLE 21 Digital expression of FUE__16 ortholog group: Treatmentchemical cold heat light nutrient treatment stress drought stressresponse nodulation deficiencies pathogen salinity barley 0.63 maize3.98 poplar 1.12 rice 1 0.63 2 sorghum 1.9 soybean 1 1 1 1.56 5 2.851.09 tomato 1.12 TOTAL 2 1 3.98 1 4.73 5 5.09 1.09 2

FUE_17 is a hypothetical protein displaying a digital expression inmaize and other plant species linking it to a response to drought(maize, barley and sorghum), to pathogens (rice, soybean and tomato) andto nitrogen deficiency and water-logging in barley.

FUE_30 and FUE31 were selected because of their annotation and thesignificant EST content derived from root particularly in the case ofFUE_30. FUE_30 and FUE_31 have a predicted invertase activity, an enzymewhich is known to delay senescence when expressed under asenescence-activated promoter (Balibrea Lara, et al. Plant Cell 16:1276-1287, 2004). FUE_30 can extend the stay-green characteristics ofthe plant and therefore increase the overall capacity to store nitrogenin plants for redistribution during grain filling.

FUE_32 is annotated as major intrinsic like protein and its wheatorthologue contains several sequences isolated from root. In addition,the maize gene selected shows homology to Brevis Radix. Brevis Radix isa novel regulator of cell proliferation and elongation that wheninactive it creates plants with shorter roots (Mouchel at al., Genes andDevelopment Vol. 18:700-714, 2004).

FUE_54 displays homology to an Auxin responsive protein. The genescontain a significant EST annotation from roots and stressed tissues, aswell as its best orthologue from sorghum and rice that are speciallyexpressed under stress conditions and probably enriched in the root.

FUE_33 and FUE_100 encode for nitrilases. Nitrilase is an importantenzyme in the auxin biosynthetic pathway that converts3-indoleacetonitrile to 3-indole-acetic acid (auxin). Increasedexpression of Nitrilases in the root will enlarge the overall auxinlevels and induce root elongation, secondary root formation, enhancedgravitropic response, etc.

FUE_34 and FUE_35 as well as FUE_46 and FUE_47 are probabletranscription factors both expressed particularly in stressed roots asjudged by the EST libraries providing the sequences of the genes. Asthese transcription factors are highly associated with water stressedlibraries it may occur that modifying the expression of these genes willactivate the genes that induce the structural and metabolic changesassociated with stresses.

FUE_36, FUE_37 and FUE_38 show some degree of similarity toABA-responsive genes and/or stress annotated genes, and have asignificant content of sequences derived from root libraries. FUE_36 hasprobably protein kinase activity with a striking similarity to a SAPK8,a protein that its expression levels are increased during ABA treatmentor under salinity conditions, demonstrating a role in the stressresponse. FUE_37 has almost all the sequences derived from root andparticularly root under water stress (as shown in Tables 22 and 23). Theorthologues from barley, wheat and sugar cane also support thecontention that the gene is mainly expressed in roots. There areindications that this gene is implicated in the response to water stressdeprivation, hyperosmotic salinity response, cold and other stresses.

TABLE 22 Digital expression of FUE_37 ortholog group: Anatomy seed-callus fruit leaf mix root ling stem xylem barley 0.35 0.84 0.24 grape2.37 maize 0.26 0.84 poplar 0.09 7 rice 0.26 0.34 soybean 0.29 0.2  0.310.21 1 TOTAL 0.55 2.37 0.99 0.57 1.9  0.24 1 7

TABLE 23 Digital expression of FUE_37 ortholog group: Treatment hormonedrought treatment mix nodulation pathogen salinity barley 1.19 0.14maize 0.4 1 rice 2.38 0.36 soybean 0.4 0.69 1 TOTAL 4.37 0.36 0.69 10.14 1

FUE_38 is derived from various stressed root libraries (as shown inTables 24 and 25) and displays a notorious similarity to PKABA1. PKABA1transcript levels are barely detectable in growing seedlings but areinduced dramatically when plants are subjected to dehydration, cold andosmotic stress (Holappa L D and Walker-Simmons M K Plant Physiol. Vol.108:1203-1210, 1995). A modified expression pattern for this gene canprovide increased abiotic stress tolerance, increased vigor, increasedyield and better use efficiency of fertilizers.

TABLE 24 Digital expression of FUE_38 ortholog group: Anatomy SecondaryESTs in ESTs in Expected Keyword Keyword Gene Production ESTs Foldp-value cell culture 3 16132 1 3 0.0367348 flower 1 89278 4.141730.241445 0.990968 seedling + 1 9012 1 1 0.344935 female flower grain 173038 3.38834 0.29513 0.976577 endosperm 1 25774 1.19569 0.8363360.709829 mix 2 90046 4.17736 0.478771 0.944464 root primary root 1033886 1.57202 6.36125 5.83E−07 system seedling 1 32466 1.50614 0.6639480.792566 seedling + 1 9012 1 1 0.344935 female flower

TABLE 25 Digital expression of FUE_38 ortholog group: Treatment hormonelight mechanical drought treatment response treatment mix nodulationpathogen salinity barley 0.26 0.93 maize 2.3 0.45 0.93 rice 0.65 0.13sorghum 0.26 0.46 2 0.93 soybean 0.26 2.75 0.14 TOTAL 3.07 0.46 0.65 20.45 2.75 0.27 2.8

FUE_48 was selected using microarray Arabidopsis expression dataavailable at (Hypertext Transfer Protocol://affymetrix (dot) arabidopsis(dot) info/narrays/supersearch (dot) pl?searchterms=afgn). Genes withenriched expression in roots and highly responsive to ABA and abioticstresses (salinity, osmoticum and cold) were identified. One of thegenes had an orthologue in maize (FUE_48) that showed enhanced contentof root-derived sequences. The probable sorghum orthologue is a genemainly composed by stress-derived sequences, particularly from waterstress and drought conditions (as shown in Table 26). FUE_48 showssimilarity to SIP (Seed Imbibition Protein).

TABLE 26 Digital expression of FUE_48 ortholog group: Treatment heathormone light nutrient drought stress treatment response nematodedeficiencies pathogen salinity barley 0.98 cotton 0.09 maize 0.73 0.18poplar 0.11 0.23 0.25 rice 0.51 0.19 0.27 0.53 sorghum 3.20 0.71 0.040.15 soybean 0.41 1.78 0.40 0.16 0.18 tomato 0.12 0.13 TOTAL 5.42 0.710.27 0.75 1.78 0.76 0.77 0.89

FUE_39 is a root-expressed calcineurin B-like Ca⁺⁺ binding protein.Calcineurins are Ca⁺⁺ sensing protein kinases differentially regulatedby stress conditions such as salt, drought, cold, and wounding stress.Calcium is involved in several root important behaviors such asgravitropism, hydrotropism, etc and is an important second messenger forvarious signal transduction pathways. Continuous activation of theprotein cascade in which FUE_39 is involved can provide advantageousproperties or desired traits compared to a reference plant, includingimproved abiotic stress tolerance, increased vigor and yield, improveduse of fertilizer, etc.

FUE_40 and FUE_41 are genes expressed in roots particularly at stressconditions. The sorghum orthologue of FUE_40 has several sequencesderived from stressed tissues including roots. FUE_41 and FUE_40 showsimilarity to stress-responsive proteins.

FUE_43, FUE_44 and FUE_45 have a distant similarity to an E. coliuniversal stress protein and their composing sequences contain asignificant fraction of root stressed tissues (as shown in Table 27 forFUE_43 and in Table 28 for FUE_44). FUE_43 shows similarity to anethylene-responsive protein. FUE_44 and FUE_45 show a weak similarity toan early nodulin-like protein. Those proteins expressed ectopicallyunder a different promoter are likely to produce favorable effects inthe transgenic plants such as enhanced stress tolerance, improved growthat optimal and adverse conditions mainly due to probable modificationsin the root architecture.

TABLE 27 Digital expression of FUE_43 ortholog group: Treatment Controlhormone light nutrient Library drought treatment response mix nodulationdeficiencies pathogen salinity barley 1.00 0.33 0.09 maize 1.42 1.550.83 rice 0.83 1.64 sorghum 1.60 0.91 0.63 soybean 0.43 0.81 0.22 tomato0.10 TOTAL 1.00 3.86 0.43 2.88 1.55 0.81 0.63 0.41 0.83

TABLE 28 Digital expression of FUE_44 ortholog group: Treatment chemicallight nutrient treatment drought response nematode deficiencies pathogensalinity waterlogged barley 0.66 0.28 grape 0.07 maize 0.92 1.69 poplar0.52 0.20 rice 0.50 0.07 sorghum 0.66 0.50 soybean 3.00 0.44 0.33 2.001.30 0.08 0.85 5.00 tomato TOTAL 3.00 2.67 1.34 2.00 1.82 0.69 2.54 5.00

FUE_50 was selected using microarray Arabidopsis expression data inwhich root specific genes were selected if they were also auxin andethylene responsive. According to the calculated digital expression, themaize orthologue presented in this invention is expressed in roots underwater stress conditions and in pathogenesis related tissues. The maizegene shows similarity to nodulin, a protein known to be involved duringnodule and lateral root development (Papadopoulou et al. Plant Mol Biol.1996; 30:403-17).

FUE_501 is a putative Arabidopsis thaliana amino acid transporter thatshows increased expression under salinity, osmoticum and is alsoAbscisic acid responsive (ABA—abiotic stress response hormone). FUE_51,a maize homologue of FUE_501 is composed mainly by sequences derivedfrom stressed root sequences and has a tentative annotation as an aminoacid transporter. The sorghum orthologue of FUE_51 is involved in stressresponses according to the digital expression. Its barley orthologue hassequences derived from roots and stressed roots. Also the wheatorthologue shows several sequences derived from root.

FUE_502 is a putative amino acid transporter which shows increasedexpression during salinity stress and osmoticum both in roots andshoots. FUE_52, its closest maize orthologue, has also similarity toamino acid transport proteins and has a significant representation ofroot and stressed root derived sequences.

FUE_503 has an impressive expression in seeds suggesting a strong rolein uptake of amino acids into the developing seeds. In addition the geneis strongly upregulated under salinity and in response to methyljasmonate. FUE_504 was chosen because of its strong response to ABA, tosalinity (specially of the shoot), to osmoticum, and cold. FUE_49 showsa strong similarity at the level of translated amino acids to FUE_504but low at the level of coding nucleotides. Its sorghum orthologue hassequences significantly derived from water stress/drought libraries.

FUE_53 is a putative amino acid transporter with almost of all thesequences composing the genes derived from root and particularlystressed roots. The sorghum, rice and wheat orthologues contain severalsequences derived from root and root under stress libraries.

FUE_101 and FUE_102 show similarity to amino acid transporters. FUE_102is specifically composed from sequences derived from cold treatedseedlings and pathogen infected corn ear tip.

FUE_55 is a gene with a striking similarity to tRNAisopentenyltransferase, an important enzyme in the cytokinin metabolicpathway. Increased expression of FUE_55 in the roots or shoots willincrease the levels of cytokinin leading to enhanced leaf expansion andcell duplication, delayed senescence, increased sink strength of thetissues, enhanced nitrogen utilization, etc. FUE_55 is particularlyexpressed in the endosperm, a tissue that is probably the strongest sinkin the plant.

FUE_505 is a putative AP2 domain-containing transcription factor presentin two important list of genes. One list contains the genes that areZeatin responsive (Zeatin is a naturally occurring cytokinin) while thesecond list of genes contains nitrogen induced genes after nitrogenaddition to nitrogen starved plants (Wang et al., Plant Physiology, June2003, Vol. 132, pp. 556-567). FUE_505 was found in the cluster of theARR (Arabidopsis response regulators) which mediate the response of theshoot to cytokinin. Root cytokinin production increase upon nitrogenavailability (Yamada et al. FEBS Lett. Vol. 436, pp: 76-80, 1998). Itsco-regulation together with ARRs indicates a possible role in thecytokinin response. It is highly likely that constitutive expression ofFUE_505 will improve nitrogen utilization and plant growth due tocontinuous activation of the cytokinin response of the shoot.

Example 2 mRNA Expression of in-Silico Expressed Polynucleotides

mRNA levels are determined using reverse transcription assay followed byquantitative Real-Time PCR (qRT-PCR) analysis. RNA levels are comparedbetween different tissues, developmental stages, growing conditionsand/or different genetic backgrounds. A correlation analysis betweenmRNA levels in different experimental conditions/genetic backgrounds asevidence for the role of the gene in the plant.

Methods

RT-PCR analysis—Root and leaves were excised fresh from maize plantsgrown on 10 liter white buckets filled with Vermiculite Size 3. Bucketswere watered with tap water until seeds from a commercial hybridgerminated. During the entire growth period (5 weeks), the plants wereirrigated with 2 liter/bucket/day with a solution pH 5.7-5.8 containing2 mM CaCl₂, 1 mM MgSO₄, 1 mM KH₂PO₄, 7 mM KCl and microelementscocktail. Ammonium nitrate was added at the following concentrations: 5mM, 0.5 mM, 0.05 mM, 0.005 mM or not at all. For experiments withPotassium nitrate instead of ammonium nitrate, the concentrations wereas follow 2 mM CaCl₂), 2 mM MgSO₄, 1 mM KH₂PO₄, 5 mM KCl, and either oneof the following concentrations of KNO₃: 5 mM, 1 mM, 0.1 mM, 0.01 mM,maintained to a final concentration of potassium of 10 mM withsupplemental concentrations of KCl.

Quantitative Real Time RT-PCR (qRT-PCR)—To verify the levels ofexpression, specificity and trait-association, Reverse Transcriptionfollowed by quantitative Real-Time PCR (qRTPCR) was effected on totalRNA extracted from several parts of the plant including for examplemature and juvenile leaves, roots and root meristems, husks, tassels,silks, etc. from plants grown on soil or pots under optimal or nutrientdeficient conditions as described above. Messenger RNA (mRNA) levels aremeasured for all the genes, previously predicted bioinformatically to beassociated with Fertilizer Use Efficiency and correlation betweenexpression levels and the plant nutrient status was analysed. Total RNAwas extracted from leaves or roots of maize, using RNeasy plant mini kit(Qiagen, Germany) using the protocol provided by the manufacturer.Reverse transcription was performed using 1.5 μg total RNA, using 300 USuper Script II Reverse Transcriptase enzyme (Invitrogen), 225 ng randomdeoxynucleotide hexamers (Invitrogen), 500 μM dNTPs mix (Takara, Japan),0.2 volume of ×5 RT buffer (Invitrogen), 0.01M DTT, 60 U RNAsin(Promega), DEPC treated DDW was added up to 37.5 μl. RT reactions wereincubated for 50 min at 42° C., followed by 70° C. for 15 min. cDNA wasdiluted 1:20 in Tris EDTA, pH=8. 5 mL of the diluted cDNA was used forqRT-PCR.

Quantitative RT-PCR was performed on cDNA (5 μL), using ×1 SYBR GREENPCR master mix (Applied Biosystems), forward and reverse primers 0.3 μM□each, and DDW was added up to 20 μL. qPCR reaction was performed in aStratagene MX 3000 real-time PCR machine with the following conditions50° C. for 2 min, 95° C. for 10 min, 40 times of 95° C. for 15 sec and 1min at 60° C., followed by 95° C. for 15 sec, 60° C. for 60 sec, and 70times of 60° C. for 10 sec+0.5° C. increase in each cycle. For eachgene, a standard curve is prepared from a pool of RTs from all samples,in 5 dilutions (dilutions—1:60, 1:200, 1:600, 1:2000, 1:10000). Thestandard curve plot [ct (cycle threshold) vs. log (concentration)]should have R>=0.98 with an efficiency in the range of 100%+/−5%. Thelevels of expression (Qty) measured in the qPCR were calculated usingthe efficiency (E) of the amplification reaction and the correspondingC.T. (the cycle at which the samples crossed the threshold)Qty=E^(−C.T.). The dissociation curves obtained were qualified for theabsence of non-specific PCR products or primer-dimers. Reactions wererepeated at least twice. The calculation method is based on theassumption that the efficiencies of the reactions of the GOI (gene ofinterest) and of the housekeeping genes are similar.

To normalize the expression level between the different tissues andgrowing conditions of maize plants, expression of each gene was dividedby the geometric mean of the expression of the following fourhousekeeping genes: Actin (GenBank Acc No AY107106), and RPL19 (GenBankAcc. No. AY103679), Cyclophyllin (GenBank Acc No X68678) and Elongationfactor 1 alpha (EF1A, GenBank Acc No AF136823).

TABLE 29  The following primers were used for qRT-PCR analysis: GENEPRIMER/SEQ ID NO:  Actin FORWARD PRIMER:CCTGACTGAACGCGGCTACT/SEQ ID NO: 1344 Actin Reverse primer:CAATGGTGATGACCTGTCCGT/SEQ ID NO: 1345 RPL19 Forward primer:GGAAAGGCAAAGTCTGGCTTG/SEQ ID NO: 1346 RPL19 Reverse primer:GCACGGGACCTAGAGTGAACC/SEQ ID NO: 1347 Cyclophilin ForwardGGACAGCTCTCCTAGATCTCTCTGA/SEQ ID NO: 1348 primer: Cyclophilin ReverseCTAAACCGCTAAATTAAACTGCGAG/SEQ ID NO: 1349 primer: EF1A Forward primer:GTGAACATGCTCTCCTTGCGT/SEQ ID NO: 1350 EF1A Reverse primer:ATTTGGGAGTGGTTGCATCC/SEQ ID NO: 1351 FUE_1 Forward PrimerACTTCCACTGCGCGTAGATGA/SEQ ID NO: 1352 FUE_1 Reverse PrimerACGGAATAATGACGATGAACCAC/SEQ ID NO: 1353 FUE_2 Forward PrimerCGAGCGACTCAAACCATGC/SEQ ID NO: 1354 FUE_2 Reverse PrimerTCTTGGTGATTGAATCCAGCG/SEQ ID NO: 1355 FUE_2_RT_2FGCATGGTGGGTAAGGTTTTCTT/SEQ ID NO: 1356 FUE_2_RT_2RCAATCTTGGTGATTGAATCCAGC/SEQ ID NO: 1357 FUE_3 Forward PrimerCACCATCCACGCACTAGTACGT/SEQ ID NO: 1358 FUE_3 Reverse PrimerCAGCTAGCCGCTTCGAGAGAT/SEQ ID NO: 1359 FUE_4 Forward PrimerAAGACGCCGATCGATACATTG/SEQ ID NO: 1360 FUE_4 Reverse PrimerACGTATTGAGTTCAGTTCTTCATGTGT/SEQ ID NO: 1361 FUE_5 Forward PrimerCCATTCAGAGGATATTTGGGTGA/SEQ ID NO: 1362 FUE_5 Reverse PrimerAATACTTGCGACCATGTCTATCCA/SEQ ID NO: 1363 FUE_6 Forward PrimerCCAGTCCACAATCTCCCTGCC/SEQ ID NO: 1364 FUE_6 Reverse PrimerGTCGTGCGCCTGGCCAAAG/SEQ ID NO: 1365 FUE_7 Forward PrimerCTGCGGTCAGAAATCTTATCGTC/SEQ ID NO: 1366 FUE_7 Reverse PrimerCGAACACAAAATCAGGCGTCT/SEQ ID NO: 1367 FUE_8 Forward PrimerCTTCCGGCTGGGTAAGTTTG/SEQ ID NO: 1368 FUE_8 Reverse PrimerATGGTTCAGTGCCGTCTCCT/SEQ ID NO: 1369 FUE_9 Forward PrimerAAGAAGAAGCGACACAGCTCAAC/SEQ ID NO: 1370 FUE_9 Reverse PrimerGCAGCAGTAGGATGTGCGATC/SEQ ID NO: 1371 FUE_9_RT_2FCAGCTCAACTGCTCAAGGACAG/SEQ ID NO: 1372 FUE_9_RT_2RGGCGCAGCAGTAGGATGTG/SEQ ID NO: 1373 FUE_10 Forward PrimerCGATGCCTACAAGAACTACACCG/SEQ ID NO: 1374 FUE_10 Reverse PrimerAGTGGTTCTTGTCCGTGTTGAAG/SEQ ID NO: 1375 FUE_10_RT_2FGCCGATGCCTACAAGAACTACAC/SEQ ID NO: 1376 FUE_10_RT_2RTGAAGATGAGGAAATCCCCG/SEQ ID NO: 1377 FUE_12 Forward PrimerTCCTGTTACAGATGGCATCCG/SEQ ID NO: 1378 FUE_12 Reverse PrimerTCTCTCTCCTTAGTGAAGACGTTCG/SEQ ID NO: 1379 FUE_12_RT_2FAGAATCTTATAAAGCCCTCACATCGT/SEQ ID NO: 1380 FUE_12_RT_2RGATGACACGGGCGAACAGTA/SEQ ID NO: 1381 FUE_13 Forward PrimerCCGCCACCTTTCCTACGTAC/SEQ ID NO: 1382 FUE_13 Reverse PrimerTCGGTCTATACTCGATTCGAAGC/SEQ ID NO: 1383 FUE_14 Forward PrimerTCTGTGTTGCTGATATTCACTCTCCT/SEQ ID NO: 1384 FUE_14 Reverse PrimerCCTTACTCTTCATGGTGTCTCTAGCTT/SEQ ID NO: 1385 FUE_15 Forward PrimerGGCCGCCTGTTAGGAGCTA/SEQ ID NO: 1386 FUE_15 Reverse PrimerCGCCTCACTCCTACCTTCATCT/SEQ ID NO: 1387 FUE_16 Forward PrimerAAGAGGCAGTGCTGTTTCCGT/SEQ ID NO: 1388 FUE_16 Reverse PrimerGCCAGAATCGAGCATAGACCA/SEQ ID NO: 1389 FUE_17 Forward PrimerGCATCAAAGAGTGGTCAATTCACA/SEQ ID NO: 1390 FUE_17 Reverse PrimerCACCCTCCTTTGCAGTGCA/SEQ ID NO: 1391 FUE_30 Forward Primer:CCAAGTCGTCTCTTAGTCCGGA/SEQ ID NO: 1392 FUE_30 Reverse Primer:CAGGATGCACGTCTTGCCT/SEQ ID NO: 1393 FUE_31 Forward Primer:ATGTGCAACGACCCTACTAACTCC/SEQ ID NO: 1394 FUE_31 Reverse Primer:TCAGTGTCCTCAGCGCGAT/SEQ ID NO: 1395 FUE_32 Forward Primer:CTCGAGCATCACTCAAACCACTAC/SEQ ID NO: 1396 FUE_32 Reverse Primer:GCCAGGCCCATTGAATCAC/SEQ ID NO: 1397 FUE_33 Forward Primer:CAGCGCTGTATGGTAAAGGTATTG/SEQ ID NO: 1398 FUE_33 Reverse Primer:TGTGTCATGGAGGCTTGCC/SEQ ID NO: 1399 FUE_34 Forward Primer:GCCACCAACTCCATTCAACTTT/SEQ ID NO: 1400 FUE_34 Reverse Primer:CTCTTATTCATGGGCTTAATTGCA/SEQ ID NO: 1401 FUE_35 Forward Primer:GACGCTCTGATTAGGCTAGGGAC/SEQ ID NO: 1402 FUE_35 Reverse Primer:CCCTGCTGTTACGTGGCC/SEQ ID NO: 1403 FUE_36 Forward Primer:TCAGTGAAGATGAGGCTCGCTA/SEQ ID NO: 1404 FUE_36 Reverse Primer:TCCCATCTAGAAGTGTGTTCTCCA/SEQ ID NO: 1405 FUE_37 Forward Primer:TGTGAGCATCAACTCTAACATGGA/SEQ ID NO: 1406 FUE_37 Reverse Primer:TGACATAAATTTCACACATTCACATCA/SEQ ID NO: 1407 FUE_38 Forward Primer:CTATGGAAATTTGTCACCGTGATC/SEQ ID NO: 1408 FUE_38 Reverse Primer:TCGCAAATTTTCACACGAGGT/SEQ ID NO: 1409 FUE_39 Forward Primer:CCGATGAGCTAGGAGAAGTGTTG/SEQ ID NO: 1410 FUE_39 Reverse Primer:TTTGGCCGATTGGGTATGTC/SEQ ID NO: 1411 FUE_40 Forward Primer:ATTGCAGAGAGGGAAAACGTAAGA/SEQ ID NO: 1412 FUE_40 Reverse Primer:AAATCAAACACGTCCAAGAACATC/SEQ ID NO: 1413 FUE_41 Forward Primer:AAATATTTGTCCTATGGGAATGGG/SEQ ID NO: 1414 FUE_41 Reverse Primer:AGTATAATAGTTCTGTCCAAAGTTGCGT/SEQ ID NO: 1415 FUE_42 Forward Primer:GGTTGCGCGCTCTGACTTA/SEQ ID NO: 1416 FUE_42 Reverse Primer:CCAACGACTCGCCTTCCTAAC/SEQ ID NO: 1417 FUE_43 Forward Primer:CTTTGTAAACCAATGCCATCAGTTAG/SEQ ID NO: 1418 FUE_43 Reverse Primer:GCTGGACTCTCTTCACTTCGCT/SEQ ID NO: 1419 FUE_44 Forward Primer:GCACTGATCAATGCCGACTG/SEQ ID NO: 1420 FUE_44 Reverse Primer:AAGGATTGGATCGGATACTTGTAGA/SEQ ID NO: 1421 FUE_45 Forward Primer:TTATCCATGATACCCCGCTGA/SEQ ID NO: 1422 FUE_45 Reverse Primer:GTGCTTGTTGACTTGACGACG/SEQ ID NO: 1423 FUE_46 Forward Primer:AGCAGCCGCTGTGTATAACAAG/SEQ ID NO: 1424 FUE_46 Reverse Primer:GGAAGGGAAAGAGGACGTCAA/SEQ ID NO: 1425 FUE_47 Forward Primer:TGAACCTACTCTTTGGGATAGCTGT/SEQ ID NO: 1426 FUE_47 Reverse Primer:ACGTGGAGAATTACATGCTGATAGTT/SEQ ID NO: 1427 FUE_48 Forward Primer:ACCATTGTTAGTGACTTAATTACCCTCA/SEQ ID NO: 1428 FUE_48 Reverse Primer:CGAAATCGATAGACCACACACG/SEQ ID NO: 1429 FUE_49 Forward Primer:CCCTTCTACAAGCAAATACTCACTCC/SEQ ID NO: 1430 FUE_49 Reverse Primer:GACCCTCTCATCTCTTATTGAACCA/SEQ ID NO: 1431 FUE_50 Forward Primer:GGCGATCCAACGGAGAAGTAG/SEQ ID NO: 1432 FUE_50 Reverse Primer:ACATCGGCAGCACGGAGT/SEQ ID NO: 1433 FUE_51 Forward Primer:CCGGTGCATAAGGAAATACGAG/SEQ ID NO: 1434 FUE_51 Reverse Primer:GACAGATTGAAAACTGCCCCAG/SEQ ID NO: 1435 FUE_52 Forward Primer:GTCGATCATCGCATTCTCCA/SEQ ID NO: 1436 FUE_52 Reverse Primer:TCCTTTCATCGTATCCCCCAT/SEQ ID NO: 1437 FUE_53 Forward Primer:CAGATCTTCTTCTCTCAGCTCCCT/SEQ ID NO: 1438 FUE_53 Reverse Primer:GGGCTGAATCAACGTCGACT/SEQ ID NO: 1439 FUE_54 Forward Primer:GAAGCTGTGGGTCTCAAGGG/SEQ ID NO: 1440 FUE_54 Reverse Primer:AGGTCGGCACGTACTCGG/SEQ ID NO: 1441 FUE_55 Forward Primer:TCTACATCCAGGCTCTCGTTAGC/SEQ ID NO: 1442 FUE_55 Reverse Primer:TTCATCTTCATCAGTAAGACCATCATC/SEQ ID NO: 1443

Results

Real-time RT-PCR analysis provided evidence that the in-silico selectedpolynucleotide sequences are indeed associated with Nitrogen Use.Although the majority of the genes were chosen because of theirassociation with drought, genes were found to be responsive to theNitrogen status within the plant suggesting a cross-talk between droughtand nutrient deficiency stress. Evidence that the RNA panel used in thisassay does reflect genuine changes associated to the nitrogen status canbe found FIGS. 3A-D in which known genes associated to nitrogen uptakeand assimilation show changes in their expression levels according tothe nitrogen fertilizer level used in the irrigation solution. Thegraphs represent the normalized expression levels found for each genedivided by the level of expression at the highest nitrogen fertilizerconcentration used in the irrigation solution. FIGS. 3A and 3B show theresults found for two high affinity nitrate and ammonium transporterrespectively. As expected, at higher concentrations of the substrate,the expression of the high affinity transporters is downregulated.Conversely, those high affinity transporters, obviously essential atconditions in which the substrate is scarce, are upregulated at lownitrate and ammonium concentrations (see FIGS. 3A and 3B). As expectedfor two key enzymes in the nitrogen assimilation pathway such asGlutamine Synthase 1C and Glutamine Synthase 2, their expression isupregulated under high substrate conditions as shown in FIGS. 3C and 3D,respectively. Typically, low affinity transporters are upregulated withhigh N concentration. Conversely, high affinity transporters areupregulated with low N concentration. Enzymes involved in N-assimilation(the conversion of n to amino acids) are upregulated inn the presence ofnitrogen. The genes identified in Example 1 show the distinctivenitrogen responsive behavior found for the control genes (FIGS. 3A-D).FUE_3 (as shown in FIG. 4A) shows upregulation at high substrateconcentrations as found for the key control assimilation enzymesindicating a clear association of this gene with the nitrogen status ofthe plant and hence its relation to nitrogen related-responses. The sameupregulation found for FUE_3 was found several other genes included inExample 1 such as FUE_12 (as shown in FIG. 4B), FUE_30 (as shown in FIG.4C), FUE_33 (as shown in FIG. 4D), FUE_34 (as shown in FIG. 4E), FUE_38(as shown in FIG. 4F), FUE_43 (as shown in FIG. 4G), FUE_47 (as shown inFIG. 4H), FUE_48 (as shown in FIG. 4I), FUE_49 (as shown in FIG. 4J) andFUE_52 (as shown in FIG. 4K).

Then again, included in Example 1 are genes that show a tightupregulation when nitrogen fertilizer is in short supply. This kind ofgenes is expected to be related to to pathways involved in nitrogenassimilation, storage and use under conditions of nitrogen deficiency asis the case of the high affinity nitrate and ammonium transporters(FIGS. 2C and 2D). FIG. 5A shows the results for FUE_4, a gene thatundergoes a strong upregulation at low nitrogen fertilizer availability,indicating a clear role of this gene in the plant endogenous response tonitrogen deficient conditions. Likewise, other genes in Example 1,display a similar nitrogen-responsiveness curve as FUE_4 such as FUE_10(as shown in FIG. 5B), FUE_37 (as shown in FIG. 5C), FUE_46 (as shown inFIG. 5D) and FUE_50 (as shown in FIG. 5E).

Taken together, the results presented herein clearly imply a closecorrelation between the in-silico selected genes and nitrogen associatedpathways.

Example 3 Gene Cloning and Creation of Binary Vectors for PlantExpression

Cloning Strategy

Twenty eight genes of the 50 genes selected in Example 1 and validatedin Example 2 above were cloned into binary vectors for the generation oftransgenic plants. For cloning, the full-length open reading frame (ORF)was first identified. EST clusters and in some cases mRNA sequences wereanalyzed to identify the entire open reading frame by comparing theresults of several translation algorithms to known proteins from otherplant species. In case where the entire coding sequence was not found,RACE kits from Ambion or Clontech (RACE=Rapid Access to cDNA Ends) wereused to prepare RNA from the plant samples described in Example 2 aboveto thereby access the full cDNA transcript of the gene.

In order to clone the full-length cDNAs, Reverse Transcription followedby PCR (RT-PCR) was performed on total RNA extracted from leaves, rootsor other plant tissues, growing under either normal or nutrientdeficient conditions. Total RNA extraction, production of cDNA and PCRamplification was performed using standard protocols described elsewhere(Sambrook J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning. ALaboratory Manual., 2nd Ed. Cold Spring Harbor Laboratory Press, NewYork.) and are basic for those skilled in the art. PCR products werepurified using PCR purification kit (Qiagen) and sequencing of theamplified PCR products is performed, using ABI 377 sequencer (AppliedBiosystems).

To facilitate cloning of the cDNAs, a 8-12 bp extension was added to the5′ prime end of each primer. The primer extension includes anendonuclease restriction site. The restriction sites are selected usingtwo parameters: a. The site does not exist in the cDNA sequence. b. Therestriction sites in the forward and reverse primers are designed so thedigested cDNA is inserted in the sense formation into the binary vectorutilized for transformation. For instance, the pPI plasmid vector wasconstructed by inserting a synthetic poly-(A) signal sequence,originating from pGL3 basic plasmid vector (Promega, Acc. No. U47295; bp4658-4811) into the HindIII restriction site of the binary vectorpBI101.3 (Clontech, Acc. No. U12640).

PCR products were purified (PCR Purification Kit, Qiagen, Germany) anddigested with the restriction sites according to the primers used(Roche, Switzerland). The digested PCR products were cloned into thebinary vector pPI, which was digested with the same restriction enzymes.The digested PCR product and the linearized plasmid vector were ligatedusing T4 DNA ligase enzyme (Roche, Switzerland). The following geneswere cloned from RNA extracted from the tissues described above usingthe following primers:

TABLE 30 genes cloned from cDNA libraries and the primers used for the cloningRestriction Enzymes Gene used for Name cloningPrimers used for amplification/SEQ ID NO: FUE_501 SmaI,Fwd-AACCCGGGAACAATGACACCACAGATCAAAACTCA/ EcoRV SEQ ID NO: 1444Rev-AGGATATCATATGAATGCTACGGCAAGC/SEQ ID NO: 1445 FUE_502 SmaI,Fwd- AAACCCGGGAACAATGAGGTACGATCAGGAAGCGG/ SacI SEQ ID NO: 1446Rev- TTGAGCTCGTTCGCTGGAGGTAACAATG/ SEQ ID NO: 1447 FUE_503 SmaI,Fwd-AACCCGGGAACAATGGATCAAAAAGTTAGACAGTT/ EcoRV SEQ ID NO: 1448Rev- TTGATATCCACAAAACTCTCTAGCTCCTGAC/SEQ ID NO: 1449 FUE_504 XbaI,Fwd-AAACCCGGGAACAATGGAGGACAAGAACAATGATAAG/ SacI SEQ ID NO: 1450Rev-TTGAGCTCGGGTCTCTTTGTCATGGAGTTC/ SEQ ID NO: 1451 FUE_33 SmaI,Fwd- CTCCCGGGAACAATGGCTCTCGTGACCTCGG/ SacI SEQ ID NO: 1452Rev- TTGAGCTCCGCAACACAGTTTCATGACCA/ SEQ ID NO: 1453 FUE_34_ XbaI,Fwd- GGTCTAGAAACAATGGGGAGGGGAAAGAT/ Evo EcoRV SEQ ID NO: 1454Rev-CGGGATATCATAAGACGCCAGTTTTACTCTTATTCATGGGC/ SEQ ID NO: 1455 FUE_34_XbaI, Fwd-GGTCTAGAAACAATGGGGAGGGGAAAGAT/ Pat EcoRV SEQ ID NO: 1456Rev-CGGGATATCATAAGACGCCAGTTTTACTCTTATTCATGGGC/ SEQ ID NO: 1457 FUE_39XbaI, Fwd - AATCTAGAATGGTGGACTTTGTTCGACGG/SEQ SacI ID NO: 1458Rev - CTGAGCTCACAGGAGGACATGCCACC/SEQ ID NO: 1459 FUE_43 XbaI,Fwd- CATCTAGAAACAATGGCGGACTCGTCGGCG/SEQ SacI ID NO: 1460Rev- TTGAGCTCGCAGTGTGCAAGAGTTCCCTC/SEQ ID NO: 1461 FUE_50 XbaI,Fwd- AATCTAGAGTAATAAGCCAACAAACAAACCG/SEQ EcoRV ID NO: 1462Rev-CAGATATCCTAGCATACCGACCCTGAAAACAC/ SEQ ID NO: 1463

Synthetic sequences of some of the cloned polynucleotides were orderedfrom a commercial supplier (GeneArt, GmbH). The synthetic DNA wasdesigned in silico, based on the putative encoded polypeptide sequencesdescribed in Example 1.

To optimize the coding sequence, codon-usage tables calculated fromplant transcriptomes were used (example of such tables can be found inthe Codon Usage Database available online at Hypertext TransferProtocol://World Wide Web (dot) kazusa (dot) or (dot) jp/codon/). Theoptimized coding sequences were designed in a way that no changes areintroduced in the encoded amino acid sequence while using codonspreferred for expression in dicotyledonous plants mainly tomato andArabidopsis; and monocotyledonous plants such as maize. Such optimizedsequences promote better translation rate and therefore higher proteinexpression levels. To the optimized sequences flanking additional uniquerestriction enzymes sites were added-SalI, XbaI, BamHI, SmaI at the 5′end and SacI at the 3′ end. The genes for which codon optimizedsynthetic sequences were prepared were: FUE_2 (SEQ ID NO: 1317), FUE_3,(SEQ ID NO: 1319) FUE_4 (SEQ ID NO: 1320), FUE_40 (SEQ ID NO: 1321),FUE_7 (SEQ ID NO: 1322), FUE_8 (SEQ ID NO: 1324), FUE_9 (SEQ ID NO:1325), FUE_10 (SEQ ID NO: 1326), FUE_12 (SEQ ID NO: 1327), FUE_13 (SEQID NO: 1328), FUE_14 (SEQ ID NO: 1329), FUE_16 (SEQ ID NO: 1330), FUE_37(SEQ ID NO: 1331), FUE_41 (SEQ ID NO: 1332), FUE_46 (SEQ ID NO: 1333),FUE_47 (SEQ ID NO: 1334), FUE_49 (SEQ ID NO: 1335), FUE_52 (SEQ ID NO:1336).

Two non-optimized sequences, designated “FUE_2_Original” (SEQ ID NO:1318) and “FUE_7_Original” (SEQ ID NO: 1323), identical to theirendogenous maize sequences were synthesized and cloned foroverexpression in transgenic plant creation.

Generation of binary vectors comprising FUE genes and plant functionalpromoters for driving expression of same—The plasmid pPI was constructedby inserting a synthetic poly-(A) signal sequence, originating from pGL3basic plasmid vector (Promega, Acc No U47295; bp 4658-4811) into theHindIII restriction site of the binary vector pBI101.3 (Clontech, Acc.No. U12640). In some cases the backbone binary plasmid used was pGIwhich is similar to pPI but the GUS gene was replaced by the GUS-Introngene (Vancanneyt. G, et al MGG 220, 245-50, 1990). pGI was used to cloneall the polynucleotide sequences, initially under the control of 35Spromoter [Odell, J T, et al. Nature 313, 810-812 (28 Feb. 1985); SEQ IDNO: 1337].

Some polynucleotide sequences were cloned under other preferentialpromoters as described below. One of the promoters, named here “RootP”(SEQ ID NO: 1338) because of its enriched expression in roots, wasamplified and cloned from genomic DNA isolated from Arabidopsis thalianaby direct PCR using the following primers.

TABLE 31  Primer name Primer sequence/SEQ ID NO: Root_P ForwardTATGTCGACTCAGATGAAGAAGGCGGCT/ (Enzyme SalI) SEQ ID NO: 1464Root_P Reverse GTCCCGGGTTTATTAGATCTCTCTAGGAG (Enzyne SmaI)TTTTGA/SEQ ID NO: 1465

The promoter is the 1110 bp upstream region of the gene ATXTH19(AT4G30290, Xyloglucan endotransglucosylase/hydrolase 19). The sequencecloned was already shown to drive root-specific expression (VissenbergK, et al. Plant Cell Physiol. 2005 January; 46(1):192-200). Thefollowing genes were cloned downstream of the RootP promoter sequence:FUE_3, FUE_4, FUE_8, FUE_9, FUE_13, FUE_14, FUE_16, FUE_33, FUE_34_Evo.For control purposes, the β-glucuronidase enzyme (GUS) encoded by theuid A gene (GENBANK ACCESSION NO: S69414).

The TT105 promoter (SEQ ID NO: 1339; 2004/081173) was used to mediateconstitutive ubiquitous expression at levels different from thosemediated by the 35S promoter. The promoter TT105 contains the upstreamregion of the ELP gene (Ectopic deposition of Lignin, GENBANK ACCESSIONNO: NM_100466, AT1G05850) including the first exon and part of the firstintron. The genes cloned under the TT105 promoter were FUE_502, FUE_504,FUE_39 and FUE_52. Here again the β-glucuronidase enzyme (GUS) encodedby the uid A gene was cloned under the TT105 promoter.

Example 4 Generation of Transgenic Plants Expressing the FUE Genes

Materials and Methods

Arabidoposis transformation—was effected according to Clough S J, Bent AF. (1998) Floral dip: a simplified method for Agrobacterium-mediatedtransformation of Arabidopsis thaliana. Plant J. 16(6): 735-43; andDesfeux C, Clough S J, Bent A F. (20000 Female reproductive tissues arethe primary targets of Agrobacterium-mediated transformation by theArabidopsis floral-dip method. Plant Physiol. 123(3): 895-904.).Briefly—Arabidopsis thaliana var Columbia (To plants) were transformedusing the Floral Dip procedure described by Clough S J and Bent A F (10)and by Desfeux C et al. (11), with minor modifications. Briefly,Arabidopsis thaliana Columbia (Col0) To Plants were sown in 250 ml potsfilled with wet peat-based growth mix. The pots were covered withaluminum foil and a plastic dome, kept at 4° C. for 3-4 days, thenuncovered and incubated in a growth chamber at 18-24° C. under 16/8 hrlight/dark cycles. The T₀ plants were ready for transformation six daysprior to anthesis. Single colonies of Agrobacterium carrying the binaryvectors harboring the FUE genes were cultured in LB medium supplementedwith kanamycin (50 mg/L) and gentamycin (50 mg/L). The cultures wereincubated at 28° C. for 48 hrs under vigorous shaking and centrifuged at4000 rpm for 5 minutes. The pellets comprising Agrobacterium cells wereresuspended in a transformation medium which contained half-strength(2.15 g/L) Murashige-Skoog (Duchefa); 0.044 μM benzylamino purine(Sigma); 112 μg/L B5 Gambourg vitamins (Sigma); 5% sucrose; and 0.2 ml/LSilwet L-77 (OSI Specialists, CT) in double-distilled water, at pH of5.7.

Transformation of T₀ plants was performed by inverting each plant intoan Agrobacterium suspension such that the flowering stem was submergedfor 3-5 seconds. Each inoculated To plant was immediately placed in aplastic tray, then covered with clear plastic dome to maintain humidityand kept in the dark at room temperature for 18 hrs to facilitateinfection and transformation. Transformed (transgenic) plants are thenuncovered and transferred to a greenhouse for recovery and maturation.The transgenic To plants were grown in the greenhouse for 3-5 weeksuntil siliques were brown and dry, then seeds were harvested from plantsand kept at room temperature until sowing.

For generating T₁ and T₂ transgenic plants harboring the genes, seedscollected from transgenic To plants were surface-sterilized by soakingin 70% ethanol for 1 minute, followed by soaking in 5% sodiumhypochlorite and 0.05% Triton X-100 for 5 minutes. Thesurface-sterilized seeds were thoroughly washed in sterile distilledwater then placed on culture plates containing half-strengthMurashige-Skoog (Duchefa); 2% sucrose; 0.8% plant agar; 50 mM kanamycin;and 200 mM carbenicylin (Duchefa). The culture plates were incubated at4° C. for 48 hours then transferred to a growth room at 25° C. for anadditional week of incubation. Vital T₁ Arabidopsis plants weretransferred to fresh culture plates for another week of incubation.Following incubation the T₁ plants were removed from culture plates andplanted in growth mix contained in 250 ml pots. The transgenic plantswere allowed to grow in a greenhouse to maturity. Seeds harvested fromT₁ plants were cultured and grown to maturity as T₂ plants under thesame conditions as used for culturing and growing the T₁ plants. Atleast 10 independent transformation events were created from eachconstruct for which bulk of T2 seeds were collected.

Maize transformation—This procedure is done according to (Bronwyn R.Frame, et al, Agrobacterium tumefaciens-Mediated Transformation of MaizeEmbryos Using a Standard Binary Vector System, Plant Physiology, May2002, Vol. 129, pp. 13-22) or to the several protocols availableelsewhere from the Plant Transformation Facility at Iowa StateUniversity (Hypertext Transfer Protocol://World Wide Web (dot) agron(dot) iastate (dot) edu/ptf/Web/mainframe (dot) htm).

Example 5 Selection of Transgenic Arabidopsis Plants Expressing the FUEGenes According to Expression Level

Materials and Methods

Transgenic plants were grown as detailed in Example 4 above. Total RNAwas extracted from 10 freshly excised flowers from the transgenic plantsas detailed in Example 2 above. In order to determine the relativeexpression level of the transgene qRT-PCR reactions were performed asdescribed in Example 2 above and the results were normalized against thegeometric mean of the expression of two housekeeping genes measured inthe same samples.

TABLE 31  Primers used for selection of transgenic plants PRIMER NAMEPRIMER SEQUENCE/SEQ ID NO:  NUE10_RT_F CCACTCTGTGGTTCAGACTAGGAAC/SEQ IDNO: 1466 NUE10_RT_R CATTCAACAGTATCATCTGGCCC/SEQ ID NO: 1467 NUE16_RT_FCAGGATGGTCTAGTGGAGTGGAT/SEQ ID NO: 1468 NUE16_RT_RGCAGAGTACTGGAACACAAGGTTATC/SEQ ID NO: 1469 NUE41_RT_FCGTAAGAGAGTGGGACTTACTGGAT/SEQ ID NO: 1470 NUE41_RT_RCCATGCCTATCAGAGTACCTGTGA/SEQ ID NO: 1471 FUE501_RT_FGTCCTGCGTAGACGAGTCGAA/SEQ ID NO: 1472 FUE501_RT_RAGCTTATGACAACAAATAGAACCGC/SEQ ID NO: 1473 NUE50_RT_FGCCACCATTGTACTCGTCCC/SEQ ID NO: 1474 NUE50_RT_RGAAGCCCGTCAGTCCAAGG/SEQ ID NO: 1475 NUE50_RT2_FCCAGTGCATGTACTACCAAGGG/SEQ ID NO: 1476 NUE50_RT2_RCTAGGCCGACTGATGCCG/SEQ ID NO: 1477 FUE501_RT2_FACTTACGCCGGAGTTATGAAAGAG/SEQ ID NO: 1478 FUE501_RT2_RAAAAGTAGCAACCATCGTAGCAATC/SEQ ID NO: 1479 FUE502_RT_751FCTAGAGGCTAAATACAAAGACAAGTTCG/SEQ ID NO: 1480 FUE502_RT_831RGAACGCACCGTACATGATCG/SEQ ID NO: 1481 FUE503_RT_349FTTATCTTTATCTACTGGTGCGTTATTGC/SEQ ID NO: 1482 FUE503_RT_430RACGTCGAATGAGGCTTACACG/SEQ ID NO: 1483 FUE504_RT_296FCCAGCAGAAGTAATTTAGATGTTGAGTC/SEQ ID NO: 1484 FUE504_RT_431RCCACCAATGGGCAATTCC/SEQ ID NO: 1485 NUE39_RT2_fGATAAGGAGGAATGGCGCAG/SEQ ID NO: 1486 NUE39_RT2_RGCATCCTCTACTTGAGAGTTGAACAC/SEQ ID NO: 1487 NUE3_RT_FCAACTTACTCCATGCGCTGGT/SEQ ID NO: 1488 NUE3_RT_RGGCTGTTGTTCCTTAAGCCTAGAG/SEQ ID NO: 1489 NUE4_RT_FATGTTGTTTGGCCAGCAGGT/SEQ ID NO: 1490 NUE4_RT_RAAGTGGAACAGCGTGTGCTCT/SEQ ID NO: 1491 NUE49_RT_FCCACAATCTTACCGTGAGACCA/SEQ ID NO: 1492 NUE49_RT_RGTACTTAGAGGTAGATGACCCGAGGT/SEQ ID NO: 1493 NUE13_RT_FTGAGAGAATGTGGAAGGTTGCAT/SEQ ID NO: 1494 NUE13_RT_RTCATGGTAGTCACAGATCCTGGTC/SEQ ID NO: 1495 NUE2-Original_FAGTGTTCGTCCTCCCAGCAC/SEQ ID NO: 1496 NUE2-Original_RCCTGCCTTTTCTGGATGTCAAC/SEQ ID NO: 1497 NUE7_RT_FTTCTCAACAGGGTGATGATGCT/SEQ ID NO: 1498 NUE7_RT_RGTCTGAACAGTCTTGGTAACAGTCTCTT/SEQ ID NO: 1499 NUE7_Original_TTGTGTTGGGCGTGAGCA/SEQ ID NO: RT_F 1500 NUE7_Original_GGGAGGTGGAAGCAAGGC/SEQ ID NO: RT_R 1501 NUE9_RT_FAATGGCTTTCGAGGGACCA/SEQ ID NO: 1502 NUE9_RT_RGGAGAACAAAGTAGATCTGCCCA/SEQ ID NO: 1503 G3PD_RT_FCCTTGACATTGTTTCCAACGCTA/SEQ ID NO: 1504 G3PD_RT_RGACAGTGGTCATGAGTCCCTCA/SEQ ID NO: 1505 UBQ4_RT_FCTTCTGAGCTTTTGTGATGTGATCA/SEQ ID NO: 1506 UBQ4_RT_RATAAGTTTTTCCCGCAGACCG/SEQ ID NO: 1507 NUE52_RT_FAGCCAAAGACCTTCGCTAACG/SEQ ID NO: 1508 NUE52_RT_RCCAGTCCTAGAGAAAGTGTATGGGA/SEQ ID NO: 1509 GUS_RT2_FGTGTGGGTCAATAATCAGGAAGTG/SEQ ID NO: 1510 GUS_RT2_RAATAACATACGGCGTGACATCG/SEQ ID NO: 1511

Results

About twelve transgenic events were screened for each construct. Theresults were highly repetitive and showed that within 12 randomtransformation events about 20 to 40 fold differences in transgeneexpression were found. Representative examples of such findings areshown in Tables 32, 33, and 34). Based on these findings about 5different events showing expression levels higher than the median weretaken for further gene function validation (see Examples 6-9).

TABLE 32 Normalized Expression level Results found for Expressionrelative to lowest NUE 43 level expression level found FUE_43 Event 10.1503277 1 FUE_43 Event 2 0.16169295 1.075603188 FUE_43 Event 30.48948083 3.256092124 FUE_43 Event 4 0.62021826 4.12577504 FUE_43 Event5 0.65818337 4.378324085 FUE_43 Event 6 0.66609819 4.430974496 FUE_43Event 7 0.77390675 5.148131499 FUE_43 Event 8 0.88588004 5.892992865FUE_43 Event 9 1.47644781 9.821528908 FUE_43 Event 10 3.3557654122.32300168

TABLE 33 Normalized Expression level Results found for Expressionrelative to lowest NUE 49 level expression level found FUE_49 Event 10.284411514 1 FUE_49 Event 2 0.40568304 1.426394573 FUE_49 Event 30.523973448 1.842307439 FUE_49 Event 4 0.606977918 2.134153816 FUE_49Event 5 0.747036597 2.626604621 FUE_49 Event 6 0.763272592 2.6836909FUE_49 Event 7 1.057302538 3.717509615 FUE_49 Event 8 1.289370084.533466532 FUE_49 Event 9 1.572327678 5.528354517 FUE_49 Event 101.750374095 6.154371426 FUE_49 Event 11 4.491236228 15.79133054 FUE_49Event 12 10.44746677 36.73362806

TABLE 34 Normalized Expression level Results found for Expressionrelative to lowest NUE 16 level expression level found FUE_16 Event 10.143683 1 FUE_16 Event 2 0.884513 6.156021 FUE_16 Event 3 0.9756266.790146 FUE_16 Event 4 1.05558 7.346604 FUE_16 Event 5 1.2960679.020344 FUE_16 Event 6 1.550371 10.79025 FUE_16 Event 7 2.16157415.04409 FUE_16 Event 8 2.918563 20.31257 FUE_16 Event 9 3.12694921.76289 FUE_16 Event 10 3.149679 21.92108 FUE_16 Event 11 5.04168135.089

Example 6 Improved Fertilizer Use Efficiency in Tissue Culture Assay

Materials and Methods

Assay 1: Nitrogen Use efficiency assay using plantlets—The assay wasdone according to Yanagisawa-S. et al. with minor modifications(“Metabolic engineering with Dof1 transcription factor in plants:Improved nitrogen assimilation and growth under low-nitrogen conditions”Proc. Natl. Acad. Sci. USA 101, 7833-7838).

Transgenic plants which were grown for 7-10 days in 0.5×MS[Murashige-Skoog] supplemented with a selection agent were transferredto two nitrogen-limiting conditions: MS media in which the combinednitrogen concentration (NH₄NO₃ and KNO₃) was 0.2 mM or 0.05 mM. Plantswere allowed to grow for additional 30-40 days and then photographed,individually removed from the Agar (the shoot without the roots) andimmediately weighed (fresh weight) for later statistical analysis.Constructs for which only T1 seeds were available were sown on selectivemedia and at least 25 seedlings (each one representing an independenttransformation event) were carefully transferred to thenitrogen-limiting media. For constructs for which T2 seeds wereavailable, different transformation events were analyzed. Usually, 25randomly selected plants from each event were transferred to thenitrogen-limiting media allowed to grow for 3-4 additional weeks andindividually weighed at the end of that period. Transgenic plants werecompared to control plants grown in parallel under the same conditions.Mock-transgenic plants expressing the uidA reporter gene (GUS) under thesame promoter were used as control.

Statistical analyses—To identify genes conferring significantly improvednitrogen use efficiency (or tolerance to abiotic stresses or enlargedroot architecture, see below) the results obtained from the transgenicplants were compared to those obtained from control plants. Plant areadata, seed weight data and plant weight data were analyzed using one-wayANOVA. To identify outperforming genes and constructs, results from theindependent transformation events tested were analyzed separately. Inaddition, genes and constructs were also analyzed taking intoconsideration the results obtained from all the independenttransformation events tested the specific construct. For gene versuscontrol analysis Student's t test or Tukey HSD test were applied, usingsignificance of p<0.05. The JMP statistics software package was used(Version 5.2.1, SAS Institute Inc., Cary, N.C., USA). The samestatistical analysis was used in Examples 6-9 which follow.

Results

Polynucleotide sequences of the present invention were assayed for anumber of commercially desired traits.

FIG. 7 shows the ability FUE_504 or FUE_39 to improve NUE of transgenicplants expressing same. Clearly the transgenic plants grown on nitrogenare bigger and heavier than control plants expressing a control geneunder the same promoter used to express the transgenes. To verify thatthe increase in the mass achieved by the transgenic plants isstatistically significant, plants were individually weighed and theresults of the entire population of events analyzed. As shown in Table35 below transgenic plants expressing FUE_504 are significantly heavierthan the control counterparts. The results demonstrate an improvement inNUE since the transgenic plants expressing FUE_504 are able to producesignificantly more biomass than control plants starting from a similarnitrogen poor-media.

TABLE 35 Nitrogen conc. Least Sq Mean weight (mg) Std Error 0.2 mMCandidate 1 5.5185185* 0.3626 0.2 mM Control 3.032 0.3769 *results aresignificantly different at □□ = 0.05

Notably, whenever T1 plants were used to assay the effect of FUE_504;therefore each plant tested is a result of a different transformationevent. Having this in mind it is clear that the effect found for T1plants is a result of the transgene which is not associated with thetransgene integration site. When T2 plants were assayed, the resultswere reproducible whereby in three independent transformation increasedfresh weight was evident under limiting nitrogen conditions. Similarresults were obtained for FUE_39 and FUE_52. As shown in Table 36 belowthree independent transgenic events contributed to the increase of theplantlet biomass under limiting nitrogen conditions. The resultsindicate that the expression of also FUE_39 and FUE_502 induces asignificant improvement in nitrogen use efficiency.

TABLE 36 Least square means of fresh weight measured from ~25 plantletsgrown for 30 days in 0.05 mM combined nitrogen concentration (NH₄NO₃ andKNO₃) (results analyzed using Student's t test) Level Least Sq MeanTT105::FUE_52 Event 1 A 6.8000000 TT105::FUE_39 Event 1 A B 6.5352941TT105::FUE_52 Event 2 B C 5.6272727 TT105::FUE_39 Event 2 B C 5.6240000TT105::FUE_504 Event 1 B C 5.5956522 TT105::FUE_39 Event 3 C D 5.4400000TT105::FUE_504 Event 2 C D 5.1666667 TT105::FUE_52 Event 3 C D E5.1473684 TT105::FUE_504 Event 3 C D E 4.8882353 TT105::GUS F G H I3.6458333 Levels not connected by same letter are significantlydifferent

Similar results were obtained when checking the same events at 0.2 mMcombined nitrogen concentration. Four events transformed withTT105::NUE_39 produced statistically significant higher biomass whencompared to control plants. Similar results were obtained with 3 eventstransformed with TT105::NUE_52 and 2 events transformed withTT105::NUE_504 as shown in Table 37.

TABLE 37 Least square means of fresh weight measured from ~25 plantletsgrown for 30 days in 0.2 mM combined nitrogen concentration (NH₄NO₃ andKNO₃) (results analyzed using Student's t test) Level Least Sq MeanTT105::FUE_504 Event 1 A 7.5250000 TT105::FUE_52 Event 1 A B 7.4571429TT105::FUE_39 Event 1 A B 7.2000000 TT105::FUE_39 Event 2 A B C6.7312500 TT105::FUE_39 Event 3 A B C D 6.6000000 TT105::FUE_52 Event 2A B C D 6.5869565 TT105::FUE_504 Event 2 B C D E 6.3280000 TT105::FUE_39Event 4 C D E F 5.8454545 TT105::GUS (CONTROL) G H I J 4.5720000 Levelsnot connected by same letter are significanty different

Positive results were also obtained in an assay performed using T1 seedsof transgenic plants overexpressing either FUE_8, FUE_14 or FUE_40 undera constitutive ubiquitous promoter (35S), as shown in Table 38 below.

TABLE 38 Least square means of fresh weight measured from ~25 plantletsgrown for 27 days in 0.05 and 0.2 mM combined nitrogen concentration(NH₄NO₃ and KNO₃) (results analyzed using Student's t test MANOVA) LeastSq Level Mean 35S::FUE_14 B 6.614423 35S::FUE_40 B C 5.552381 35S::FUE_8C 5.252 35S::GUS D E F 4.217095 Event 1 35S::GUS F 3.27 Event 2 Levelsnot connected by same letter are significantly different

Similar results were obtained for different events of other constructsexpressing FUE genes. An additional construct bearing the FUE_39 geneunder the 35S promoter was checked using this assay as shown in theTable 39 below. In addition, two independent transformation eventsoverexpressing the FUE_49 gene and additional events transformed witheither FUE_4, FUE_3, or FUE_43 showed significantly higher biomassproduced at both nitrogen limiting conditions tested as shown in table39 below.

TABLE 39 Least square means of fresh weight measured from ~25 plantletsgrown for 27 days in 0.05 and 0.2 mM combined nitrogen concentration(NH₄NO₃ and KNO₃) (results analyzed using Student's t test MANOVA) LeastLevel Sq Mean 35S::FUE_43 event 1 A 5.572 35S::FUE_49 event 1 A B 5.02535S::FUE_3 event 1 A B 4.924 5134.1 A B C 4.7 35S::FUE_49 event 2 A B CD 4.512 35S::FUE_4 event 1 A B C D E 4.383333 wild type F G H I J K L3.123944 35S::GUS H I J K L M N O P 2.702041 Levels not connected bysame letter are significantly different

Taking into account the results obtained using this assay, several FUEgenes have shown their ability to induce a significant improvement innitrogen use efficiency. The genes that showed significant results are:FUE_504, FUE_39, FUE_52, FUE_502, as well as the following group ofgenes: FUE_14, FUE_8, FUE_40 and FUE_49, FUE_3, FUE_4 and FUE_43.

Assay 2: Nitrogen Use efficiency whole plant assay: Growth rate atlimited nitrogen concentration—An additional assay designed to identifyimproved nitrogen use efficiency was developed. This assay follows therosette area growth of plants grown in the greenhouse at limitednitrogen availability. Surface sterilized seeds were sown and plantsgrown at 25° C. under 23-hour light 1-hour dark daily cycles for 7-10days in 0.5×Murashige-Skoog medium in the presence of 2% sucrose. Thenseedlings of similar size were carefully transferred to 100 ml potsfilled with an inert growth medium support (a fine perlite mix).Seedlings were allowed to develop for a further week irrigated with asolution containing abundant supply of micro and macronutrients.Fertilizer excess was washed from the pots with at least two volumes oflow-nitrate tap water. Then, plants were individually inspected and onlyhealthy plants were chosen for growth rate analysis. The chosen plantswere randomly distributed in several trays and used to assay for growthrate analysis under nitrogen limiting conditions. Constant nitrogenlimiting conditions were achieved irrigating the plants with a solutioncontaining 0.5 mM inorganic nitrogen (combined KNO₃ and NH₄NO₃concentration), supplemented with 2 mM CaCl₂), 1.25 mM KH₂PO₄, 1.50 mMMgSO₄, 5 mM KCl, 0.01 mM H₃BO₃ and microelements. To follow plantgrowth, trays were photographed the day nitrogen limiting conditionswere initiated and subsequently every 2-3 days for ˜20 additional days.Rosette plant area was then determined from the digital pictures usingthe methodology described in FIG. 6. ImageJ software was used forquantifying the plant size from the digital pictures (Hypertext TransferProtocol://rsb (dot) info (dot) nih (dot) gov/ij/) utilizing proprietaryscripts designed to analyze the size of rosette area from individualplants as a function of time. Percentage of growth was calculated as theratio of the plant rosette area divided by the initial plant areameasured at day 1. To identify the transgenic plants within theexperiment, the presence of the selection marker gene was checked usingPCR. Leaf samples were taken from the plants, genomic DNA was extractedand served as template for a PCR using primers specific for theselection marker gene. Positive plants were tagged in order to excludefrom the analysis the non-transgenic plants.

TABLE 40  Primers used for the PCR analysis: Primer namePrimer sequence/SEQ ID NO:  NPTII-F CGAGAAAGTATCCATCATGGC/SEQ IDNO: 1512 NPTII-R ATGTCCTGATAGCGGTCCGC/SEQ ID NO: 1513

Results

In agreement with the results obtained in the previous assay in whichFUE_39 showed improved NUE, the results presented herein show thatFUE_39 significantly improved NUE in whole plants. As shown in Table 41below, two independent transgenic events expressing FUE_39 were found todisplay enhanced NUE when compared to either wild type or transgenicplants expressing the GUS reporter gene under the same promoter used toexpress the FUE_39 transgene.

TABLE 41 FUE_39 shows the results of a growth rate assay under limitingnitrogen conditions. Two events showing enhanced NUE were shown. LevelLeast Sq Mean 35S::FUE_39 A 867.53380 Event 1 35S::FUE_39 B 685.52595Event 2 Wild type C D 590.28343 Levels not connected by same letter aresignificantly different

In addition to FUE_39, also events overexpressing FUE_7 and FUE_41 werefound to display enhanced growth rate under limiting nitrogenconditions. As shown in Table 42, FUE_7, FUE_41, FUE_50, FUE_16expression under the 35S promoter are significantly better than controlplants (wild type or expressing a reporter gene under 35S promoter).

TABLE 42 Least Sq Level Mean 5351.3 A 1184.9559 (35S::FUE_7) 5375.1 A1010.5752 (35S::FUE_41) 5345.1 A B  956.9474 (35S::FUE_50) 5361.2 B C 824.0414 (35S::FUE16) 4545.7 D E F G H I  515.8663 (35S::GUS) Levelsnot connected by same letter are significantly different

Thus, the assay described here follows the ability of the plant to growat limiting nitrogen conditions. Since the transgenic plants describedabove developed significantly faster than control plants it can beassumed that the outstanding events were able to assimilate the nitrogenavailable and to convert it to organic matter. As all the plants in theexperiment were treated similarly, the results indicate that theoutstanding events were able to utilize more efficiently the nitrogenavailable and therefore display enhanced nitrogen use efficiency.

Assay 3: Seed Yield assay at limited nitrogen availability—Maize plantsabsorb and store the majority of the nitrogen in leaves and stalk untilflowering. Stored nutrients are then redistributed to the developingkernels. In the present assay plants were grown in limited nitrogenconditions as detailed in Assay 2 above for about 45 days and then grownuntil full maturity with tap water only. Plants are therefore forced toredistribute the stored nitrogen to the developing seeds. Plants thatstore more nitrogen are expected to have a better yield. Seeds werecollected from individual plants and the yield was measured as seedweight. The yield of at least about 6 transgenic plants was usuallymeasured for each event tested.

Results

In this assay transgenic plants overexpressing FUE_43 (under the 35Spromoter) were found to have increased seed yield as shown in Table 43.The results of one of the events are presented below.

TABLE 43 Level Least Sq Mean 5151.2 (35s::NUE43) A 0.10411111 4542.4(35S::GUS) B C D E F G H 0.06647500 Levels not connected by same letterare significantly different

Taken as a whole the results shown here demonstrate that genes from thisinvention are capable of improving different aspects of nitrogen useefficiency as demonstrated by the results of the different assaysdisclosed here.

Example 7 Evaluating Transgenic Plant Growth Under Abiotic StressConditions

Tolerance to salinity or osmotic stress is aimed at identifying genesthat confer better germination, seedling vigor or growth in high salt,drought or combination of same or other environmental stresses. Plantsdiffer in their tolerance to salt stress, high osmoticum and droughtduring different stages of development. Therefore seed germination,plant development, and yield were evaluated independently under thedifferent stress conditions.

A typical salinity tolerance test is produced taking plants at differentdevelopmental stages and irrigating them with increasing concentrationsof NaCl (for example 50 mM, 100 mM, 200 mM, 400 mM) or constantconcentration of NaCl. Transgenic plants are then compared to controlplants in their external phenotypic appearance, degree of wilting, andoverall success to reach maturity and yield progeny at concentrationsinhibitory to control plants. Quantitative parameters of tolerancemeasured include, the average wet and dry weight, and the weight of theseeds yielded, the average seed size and the number of seeds producedper plant. Osmotic stress assays (including NaCl and mannitol assays)are conducted to determine if an osmotic stress tolerant phenotype isNaCl-specific or if it is a general osmotic stress related phenotype.Drought assays are performed for example halting irrigation during longperiod and measuring wilting rate, recovery and final yield. Plantstolerant to osmotic stress are in general more tolerant to drought,salinity and freezing conditions and therefore are highly valuable interms of agronomic traits.

Materials and Methods

The method used to test the plants for improved abiotic stress toleranceincludes the test of germination and seedling growth under adverseconditions such as high salinity and high osmoticum.

Germination assay under abiotic stresses—Germination tests compare thepercentage of seeds from transgenic plants that could complete thegermination process (radicle protrusion from the seed coat and completeopening of the cotyledons) to the percentage of seeds from controlplants treated in the same manner. Evaluation of germination andseedling vigor was conducted for three weeks after planting. To measuregermination and seedling growth, seeds from T2 plants were surfacesterilized and individually sown on square agar plates containing forexample, solidified basal media (50% Murashige-Skoog medium(MS)+vitamins supplemented with 0.8% plant agar) supplemented with highsalinity (150 mM NaCl) or high osmoticum (210 mM mannitol). Aftersowing, plates were transferred for 2-3 days at 4° C. for stratificationand then grown for three weeks.

To follow the germination and growth at adverse conditions plants werescreened manually or automatically and plant size was determined.Various independent transformation events selected from Example 5 wereanalyzed from each construct. Plants expressing the genes from thisinvention were compared to control plants (wild type or mock-transformedplants) sown on the same plates under the same conditions.

Seedling growth under abiotic stress—A complementary experimentperformed with seedlings follows the tolerance of the plants to adverseconditions. Surface sterilized seeds were sown in basal media [50%Murashige-Skoog medium (MS)+vitamins supplemented with 0.8% plant agaras solidifying agent] in the presence of Kanamycin (for transgenicplants) or in its absence (for wild-type control plants). After sowing,plates were transferred for 2-3 days at 4° C. for stratification andthen grown at 25° C. under 23-hour light 1-hour dark daily cycles for 7to 10 days. At this time point, seedlings randomly chosen were carefullytransferred to plates containing high salinity conditions (150 mM NaCl)or conditions resembling the high osmolarity found during drought (210mM mannitol). Plant growth was followed as a function of time usingdigital imaging. To follow the plant growth at adverse conditions plantswere photographed the day they were transferred to the stress conditions(Day 1). Pictures were subsequently taken every few days aftertransferring the plants to the stress condition and up to 12 days afterthe transfer. Rosette plant area was then determined from the digitalpictures. ImageJ software was used for quantifying the plant size fromthe digital pictures (Hypertext Transfer Protocol://rsb (dot) info (dot)nih (dot) gov/ij/). Proprietary scripts were designed to analyze thesize of individual plants rosette areas as a function of time. FIG. 6shows the methodology used for image area quantification. Five to tenindependent transformation events were analyzed from each construct andat least 6 randomly selected plants from each event were analyzed ineach stress experiment. Plants expressing the genes from this inventionwere compared either to control plants sown on the same stress inducingplates (internal controls) or to the average measurement of all thecontrol plants used in the same experiment (all controls).

Results

Salinity and osmoticum tolerance were assayed by following plant growthduring stress, as evidenced by plant green area. Several genes andconstructs were checked using the procedure described above andsignificant tolerance results were found. For instance, transgenicplants overexpressing the following genes have shown statisticallysignificant improved tolerance to high salinity stress: FUE_7, FUE_10,FUE_14, FUE_16, FUE_33, FUE_37, FUE_39, FUE_40, FUE_41, FUE_47, FUE_50,FUE_502, FUE_503 and FUE_504.

In addition, the following genes provided statistically significantresults in coffering abiotic stress tolerance to the transgenic plantsoverexpressing same when exposed to high osmolarity stress: FUE_3,FUE_9, FUE_10, FUE_13, FUE_37, FUE_39, FUE_40, FUE_41, FUE_43, FUE_49,FUE_502, FUE_503 and FUE_504.

In addition, transgenic plants expressing FUE_43 and FUE_503 were foundto have increased ability to germinate under high osmoticum conditions(Data not shown).

Furthermore, transgenic plants expressing the following genes have shownsignificantly increased abiotic stress tolerance to either salinity orhigh osmolarity: FUE_10, FUE_37, FUE_39, FUE_40, FUE_41, FUE_502 andFUE_504.

It should be noted that the significance of the results is in generalsupported by several transgenic events expressing the above genes and insome cases also under different promoters. Table 44a-b summarize thesefindings. Results are expressed as the growth area percentage related tothe size of the plant when transferred to stress conditions (i.e., Day1).

TABLE 44a list of genes showing improved seedling growth under salinitystress Gene Promoter Event/Gene Least Sq Mean Salinity Seedling GrowthAssay FUE_502 35S 5125.3 A 402.61787 CONTROL B 270.40066 FUE_503 5184.3A 314.80237 CONTROL B 163.06265 FUE_504 35S 5147.5 A 335.25831 CONTROL B258.27089 Level TT105 5432.9 A 525.38863 5433.5 A 382.86771 5433.4 A346.97902 5433.6 A 342.96170 CONTROL B 257.88942 FUE_7 35S 5351.8 A508.52644 CONTROL B 410.02970 FUE_10 35S 5331.1 A 404.86015 CONTROL B320.63800 Experiment performed on T1 plants FUE_14 RootP RootP::FUE14 A239.97813 35S::GUS B 193.74654 35S 5451.3 A 170.50102 5454.1 A 154.771555454.4 A B 151.54574 CONTROL C 130.92706 Experiment performed on T1plants FUE_16 RootP RootP::FUE16 A 184.69992 35S::GUS B 146.24402Experiment performed on T1 plants FUE_33 RootP RootP::FUE33 A 237.7345535S::GUS B 202.99142 Experiment performed on T1 plants FUE_37 RootPRootP::FUE37 A 228.14620 35S::GUS B 195.15386 FUE_39 35S 5131.1 A272.46189 CONTROL B C 195.66170 Experiment performed on T1 plants FUE_4035S 35S::FUE_40 A 261.52868 35S::GUS B 160.55573 FUE_41 35S 5373.1 A452.27789 CONTROL B C 373.83060 Experiment performed on T1 plants FUE_4735S FUE47 A 202.37548 35S::GUS B 142.22673 FUE_50 5343.1 A 452.75078CONTROL B 376.63355

TABLE 44b list of genes showing improved seedling growth under osmoticumstress Gene Promoter Event/Gene Least Sq Mean Osmoticum Seedling GrowthAssay FUE_502 35S 5125.9 A 5125.10 B 156.16627 5125.1 B 145.40683CONTROL C 92.51323 Experiment performed on T1 plants TT105 TT105::FUE502A 145.94616 TT105:GUS B 129.39693 FUE_503 5182.3 A 163.93440 5181.2 A151.83208 CONTROL B 126.70438 Experiment performed on T1 plants FUE_504TT105 TT105::FUE504 A 172.68759 TT105:GUS B 145.85106 FUE_3 35S 5213.1 A309.25610 CONTROL B C 244.05079 FUE_9 RootP RootP::FUE9 A 213.0308135S::GUS B 175.62303 FUE_10 35S 5331.1 A 438.55622 CONTROL B 202.58577FUE_13 35S 5235.2, 7 A 312.47256 CONTROL, 7 B C 233.04378 Experimentperformed on T1 plants RootP RootP::FUE13 A 173.97881 35S::GUS B134.41030 Experiment performed on T1 plants FUE_37 RootP RootP::FUE37 A205.43584 35S::GUS B 168.99783 FUE_39 35S 5131.1 A 261.25201 CONTROL B177.03349 Experiment performed on T1 plants FUE_40 35S 35S::FUE_40 A142.16470 35S::GUS B 125.81481 FUE_41 35S 5373.2 A 306.74015 5371.4 B231.50773 CONTROL C 196.22318 FUE_49 35S 5243.3, 7 days A 382.37255CONTROL, 7 B C 270.81987 days

Example 8 Evaluating Changes in Root Architecture in Transgenic Plants

Many key traits in modern agriculture can be explained by changes in theroot architecture. Root size and depth correlates with drought toleranceand fertilizer use efficiency. Deeper root systems can access water instored in deeper soil layers. Similarly, a highly branched root systemprovides better coverage of the soil and therefore can effectivelyabsorb all macro and micronutrients available resulting in enhancedfertilizer use efficiency. To test whether the transgenic plants producea different root structure, plants were grown in agar plates placedvertically. Plates were photographed every few days and the maximallength and total area covered by the plant roots were assessed from thedigital pictures. From every construct created, several independenttransformation events were checked in replicates. To assess significantdifferences between root features, one ANOVA using Students t-test wasemployed in order to identify the events showing outstanding rootfeatures and to provide a statistical score to the findings (statisticalassays are described above).

Results

When analyzing different aspects of root architecture such as total rootarea covered and maximal depth (length) achieved by the root system, itwas found that transgenic plants expressing FUE genes have remarkablefeatures. For instance, transgenic plants overexpressing FUE_10 andFUE_49 were found to have a higher root area coverage based onmeasurements done 21 days following germination (see Table 45).

TABLE 45 Root Area (in cm²) measured at day 21 Level Least Sq Mean 21days 35S::NUE10 A 1.3313852 21 days 35S::NUE49 A 1.3253389 21 days35S::GUS B C 1.1289837 Levels not connected by same letter aresignificantly different

Similarly, plants overexpressing FUE_16 and FUE_7 original were found toproduce the longest root architecture (see Table 46 below). FUE_16 wasfound to produce the fastest penetrating root system from all theconstructs tested at all days tested (7, 14 and 21 days aftergermination).

In practice, these findings suggest that plants overexpressing FUE_16and FUE_7_Original reach deeper soil layers compared to other plants atthe same period of time. These plants are expected to maximally retrievefertilizers and water from the soil and withstand better longer periodsof drought.

TABLE 46 Statistical analysis of root length (in cm) measured at day 21Level Least Sq Mean 21 days 35S::FUE_7_Original A 6.5156396 21 days35S::FUE_16 A B 6.1808293 21 days 35S::GUS C D 5.5097884 Levels notconnected by same letter are significantly different

The enhanced root architecture allows transgenic plants expressing FUEgenes to absorb more successfully the nutrients (fertilizers) and wateravailable in the soil. Increased uptake and nutrient recovery is a keyfeature for improved fertilizer use efficiency and abiotic stresstolerance. Deeper root systems allow plants to access water available indeeper soil layers enhancing the tolerance to extended drought periodsand therefore ensuring high yields also under harsh conditions.

Example 9 FUE_40 Expressing Plants are Characterized by a PlantArchitecture for Maximal Seed Production

The presence of multiple seed producing organs (pods, fruits, etc) andstrong flowering stems is a key feature to produce high seed yields.While reducing the present invention to practice, the present inventorshave surprisingly found that transgenic plants expressing FUE_40 under aconstitutive promoter have improved reproductive organ architecture. Theflowering stems were unusually stiff and strong and carry a large numberof siliques (see FIG. 9 showing different independent transformationevents). The results were confirmed by a triple blind assay identifyingplants having more siliques per plant, probably due to faster growth oflateral inflorescence shoots. Interestingly, all the plants weredifferent transformation events bearing the same construct, NUE40 geneunder 35S promoter (see FIG. 9). This alteration in plant developmentcharacteristics can be used to significantly increase seed yield of aplant.

Example 10 CT-9 (SEQ ID NO: 1340, 1341) and CT-71 (SEQ ID NO: 1342,1343) Confer Drought Tolerance and Increased Yield of Transgenic Tomato

While further reducing the present invention to practice the presentinventors have surprisingly uncovered that CT_9 (SEQ ID NOs. 1340, 1341previously disclosed in PCT Application No. WO2005/121364) and CT_71(SEQ ID NOs. 1342 and 1343, previously disclosed in PCT Application No.WO2005/121364) confer drought resistance and increased yields intransgenic plants expressing same. CT_9 and CT_71 were disclosed in PCTApplication No. WO2005/121364 to Evogene Ltd. and in U.S. Pat. No.5,597,718 (CT_71).

Materials and Experimental Procedures

Drought assay—The drought assay described herein was effected in fieldconditions mimicking drought stress by controlling the amount of watersupplied and the drought intervals. The assay, one source drippingirrigation system (OSDI), is similar to the farmer field since itcreates water deficiency in a relatively uniform manner and enables tomeasure the effect of drought on small size populations of plants.

Thus, the present irrigation method may be effected as follows:

-   -   (a) placing on or in a soil a dripping irrigation system so as        to obtain irrigation holes distributed 20-40 cm (e.g., 30 cm)        from one another in the X and Y directions; and    -   (b) continuously irrigating through each of said irrigation        holes at an irrigation rate of 0.5-2 liter water per hour.

One configuration of an irrigation system (10) for mimicking droughtconditions which comprises the following components and illustrated inFIG. 10 may be used in accordance with the teachings of the presentinvention:

-   -   (i) a dripping irrigation system having a plurality of        irrigation holes (12) distributed 20-40 cm from one another in        the X and Y directions; and    -   (ii) a water supply and control system (14) for continuously        irrigating through each of said irrigation holes 0.5-2 liter        water per hour.

Preferred drippers used are pressure-compensated thick-walled dripperline with anti-siphon and anti-drain systems (UniRam™ CNL #16010, FlowRate 1.6 l/h from Netafim Israel). Anti-siphon system prevents dirtbackflow into the dripper line while anti-drain (CNL) system eliminatesdrainage and refill effect, and improves efficiency in pulse irrigation.

The OSDI method was developed on the basis of the line source sprinklersirrigation system (Hanks et al. 1976 Soil Sci. Soc Am. J. 40 p. 426-429)with significant modifications. Instead of sprinklers irrigation adripping irrigation was used. In order to create a uniform and deep wetlayer (at least 60 cm depth) and not the onion shape layer that istypically created by dripping irrigation a low pressure compensatingdripping irrigation system was used that allow providing small amount ofwater in a relatively long period of time.

The experiment was created in light soil during summer 2006 (from Julyto September), in an open field nethouse near Rehovot, Israel.

The water capacity of the soil was measured using the standardprocedures by sampling soil from the following three depths: 0 to 20 cm,20 to 40 cm and 40 to 60 cm. The water content in these soil layers wasmeasured routinely every week. The soil contained 5% hygroscopic waterwhile the maximum water capacity of the soil was 20%. All fertilizerswere applied into the soil prior to sowing. The amount of bothphosphorus and potassium was calculated to be sufficient for all season.Nitrogen was applied as recommended, equally to all the treatmentsthrough the irrigation system.

Each row, 193 cm wide, contained three dripping irrigation linescreating coverage of nine drippers per 1 sq. m. The water control wasdone separately for each treatment. The soil was dried completely beforethe beginning of the experiment.

Sowing was done in a nursery conditions under regular water irrigation.The seedlings were transplanted after four weeks into wet soil. Thewater amount that was used to uniformly irrigate before transplantingreached the maximum water capacity (20% w/w) at 60 cm depth howeverwithout creation of water overload. Each plant was transplanted near adripper, 30 cm distance between plants with a total density of 2600plants per 1000 sq. m, according to commercial growth protocol. Theexperiment was structured in four blocks containing three rows irrigatedwith different water levels and intervals (WLI-0, WLI-1, WLI-2). Thedifferent water regimes started only four weeks following transplantingwhen plants reached the flowering stage. The amount of water suppliedevery week during the assay was calculated at every beginning of theweek following the recommendations of standard growing protocols.

WLI-0 treatment received the recommended total weekly irrigation volume,but divided in three irrigations. The two other treatments (WLI-1 andWLI-2) represent two different level of water deficiency. WLI-1 was alsoirrigated three times a week, but the amount of water supplied was halfof the irrigation supplied to WLI-0. At the end of every week, WLI-1plants received the amount of water required to reach maximum soil watercapacity. WLI-2 was not irrigated during the week but rather at thebeginning of every week. Water was supplied to achieve the maximum watercapacity. The water stress experiment lasted throughout the floweringperiod (23 days) corresponding to 4 cycles of the stresses describedabove. Afterwards, all the treatments received the recommended amount ofwater.

The calculation of the water amount was equal to the difference betweenwater content in dry soil and soil with maximum water capacity. At theend of each stress cycle the water amount was compared betweentreatments according to actual water content in the soil (Table 47)

TABLE 47 Water content (%) in the soil at the end of the 4^(th) stresscycle (23 days) Water content in the soil (% w/w) Treatment/Soil depth(cm) Control - WLI-O WLI-1 WLI-2  0-20 17 14 10 20-40 16 12 12 40-60 1512 11Of note, during the stress period treatments WLI-1 and WLI-2 received intotal 75% less water compared to control (WLI-0).

Gene cloning and expression—Bioinformatic identification, cloning andphenotypic evaluation of CT-9 and CT-71 was described in Patent No:WO2005/121364.

CT-9 or CT-71 ligated to binary constructs, comprising the 35S promoterwere transformed into tomato plants via Agrobacterium tumefaciencetransformation.

60 μL of Agrobacterium tumefaciens LB4404 competent cells (about 10⁹cells/mL) were transformed with 20 ng of binary plasmid viaelectroporation, using a MicroPulser electroporator (Biorad), 0.2 cmcuvettes (Biorad) and EC-2 electroporation program (Biorad).

Agrobacterium cells were grown on 0.8 mL LB liquid medium at 28° C. for3 hrs and 0.2 mL of the cell suspension were plated on LB-agar platessupplemented with the antibiotics streptomycin 300 mg/L (forAgrobacterium strain LB4404) and kanamycin 50 mg/L (Sigma). Plates werethen incubated at 28° C. for 48 hrs. Agrobacterium colonies were grownand PCR amplification was performed on Agrobacterium cells, usingprimers which were designed to span the inserted sequence in the binaryvector.

PCR products were separated on 1.5% agarose gels and product sizes weredetermined by comparing to DNA ladder (MBI Fermentas). PCR products withthe predicted size were sequenced using the primers which were used forthe PCR amplification. Sequencing of the inserted sequence was performedto verify that the right clones were introduced into the Agrobacteriumcells.

DNA sequencing was effected using ABI 377 sequencer (AmershamBiosciences Inc.).

Transformation of Micro-Tom tomato plants with CT_9 or CT-71—Tomato(Lycopersicon esculentum, var MicroTom) transformation and cultivationof transgenic plants was effected according to Curtis et al. 1995Methods Mol Biol. 1995; 44:59-70, and Meissner et. al. 2000. Plant J.2000 May; 22(3):265-74. Following transformation, T1 MicroTom tomatoplants were grown in a mix contained in 1000 ml pots, until fruit setand T2 seeds harvesting. Transgenic micro-tom tomato plants overexpressing the CT-9 or CT-71 genes under the regulation of the 35Spromoter were cross-pollinated with M82 commercial variety plants.Taking randomly four plants originated from four different insertionevents representing the position effect. The F1 hybrids which includethe transgene were used for further evaluation. As a negative control,non-transgenic plants that segregated from the same transgenicpopulations were used. To distinguish between transgenic and nontransgenic plants a PCR screening was effected using primers of theNPTII gene as described in example 6.

Results

At the fruit ripening stage of about 90% red fruits, plants wereevaluated and fruits were harvested. Plant performance was evaluated bymeasuring mass of the canopies, root length and mass. The yield wasmeasured by taking green and red full size fruits for each plant. Theresults were analyzed using one side Anova test and summarized in Table48, below.

TABLE 48 Total fruit yield of the plant lines grown under severe waterstress (WLI-2) expressing CT-9 gene compared to control plants. Mean oftotal full size Mean of total full size Name of fruit weight per plant(g) - fruit weight per plant (g) - the line gene construct bestperforming event Control - 656 c 656 c non transgenic plants CT-71 692bc 707 bc CT-9 782 a 840 a a, b, c—Levels not connected by same letterare statistically significantly different at P < 0.05

The transgenic lines overexpressing CT-9 showed significantly higherfruit yield under severe water deficiency conditions (WLI-2) compared tocontrol plants.

The improvement in yield was observed also under regular waterirrigation both for lines expressing CT-9 and CT-71 (see Table 49,below).

TABLE 49 Total fruit yield of the lines over expressing CT-9 compared tocontrol plants grown under favorable water irrigation (WLI-0) Mean oftotal full size Mean of total full size Name of fruit weight per plant(g) - fruit weight per plant (g) - the line gene construct bestperforming event Negative  820 b  820 b control - non transgenic plantsCT-71 1010 a 1046 a CT-9  993 a  997 ab a, b, c—Levels not connected bysame letter are statistically significantly different at P < 0.05

Example 11 Plants Overexpressing FUE_34_Evo are Characterized byEnhanced Lateral Branching

Control of lateral buds activation can have an important role in modernagriculture and breeding of flowers and ornaments. In the case of modernmaize breeding, breeders worked for many years towards the reduction oftillers (lateral branches) in order to create single husk plants thatreach maturation concurrently. On the other hand, control of tilleringin rice breeding has also importance since multiple tillers will allowthe production of several panicles on the same plant while overtilleringhas deleterious effects on the plant. Similarly, lateral branching isimportant for ornamentals, for example to produce multiple flowers fromthe same stem, or to produce single large flowers from each stem.

As shown in FIG. 8 plants expressing exogenous FUE_34_Evo arecharacterized by multiple branching of the shoot. Expression of FUE_34Evo (SED ID No: 54) under a constitutive ubiquitous promoter causedunusually active lateral branching phenotype. All the transgenic eventsobtained with these constructs display this particular branchingphenotype at every node. This phenotype can be a result of the loss ofthe plant apical dominance or more likely to the continuous expressionof Evo_34_Evo under an ubiquitous constitutive promoter. Evo_34 is aMADS box transcription factor that contains in addition putativephosphorylation sites for cAMP- and cGMP-dependent protein kinase,Casein kinase II and Protein kinase C. Expression under a root preferredpromoter such as RootP caused the creation of a compact highly branchedroot phenotype. FUE_34_Evo can be therefore used for example to engineerplants with controlled branching adding a constitutive or induciblepromoter active at certain anatomical parts of the plant and/or atcertain developing stages. Similarly the gene can be silenced and inthis way tillering or lateral branching will be highly likely avoided.Another potential use for this gene is for MAS (Marker assistedbreeding).

Example 12 Homologous and Orthologous Sequences

Table 50 lists a summary of orthologous and homologous sequences of thepolynucleotide sequences and polypeptide sequences of the inventionidentified using BLAST (TBLASTX program) having at least 85% similarityon at least 85% of the entire protein length.

TABLE 50 SEQ ID NO: Gene Polynucleotide/polypeptide organism Speciescoverage percent_identity FUE_1 219/768  maize Zea mays 100 100 FUE_1220/769  rice Oryza sativa 89.47 77.44 FUE_1 221/770  sorghum Sorghumbicolor 87.22 81.95 FUE_1 222/771  sugarcane Saccharum officinarum 88.7284.96 FUE_1 223/772  wheat Triticum spp. 96.99 96.24 FUE_1 224/773 wheat Triticum spp. 100 98.5 FUE_2 225/774  maize Zea mays 98.48 95.45FUE_2 226/775  maize Zea mays 100 100 FUE_2 227/776  maize Zea mays 100100 FUE_2 228/777  sorghum Sorghum bicolor 89.39 84.85 FUE_2 229/778 sorghum Sorghum bicolor 89.39 84.85 FUE_2 230/779  sorghum Sorghumbicolor 89.39 84.85 FUE_2 231/780  wheat Triticum spp. 100 100 FUE_3232/781  maize Zea mays 92.08 88.12 FUE_3 233/782  maize Zea mays 10099.01 FUE_3 234/783  maize Zea mays 100 100 FUE_3 235/784  maize Zeamays 100 100 FUE_3 236/785  sorghum Sorghum bicolor 88.12 85.15 FUE_3237/786  sorghum Sorghum bicolor 88.12 85.15 FUE_3 238/787  sugarcaneSaccharum officinarum 96.04 91.09 FUE_3 239/788  wheat Triticum spp. 10099.01 FUE_4 240/789  maize Zea mays 97.63 96.45 FUE_5 241/790  barleyHordeum vulgare 89.68 76.98 FUE_5 242/791  barley Hordeum vulgare 89.6876.98 FUE_5 243/792  cotton Gossypiumssp. 85.71 72.22 FUE_5 244/793 cotton Gossypiumssp. 85.71 72.22 FUE_5 245/794  maize Zea mays 91.2777.78 FUE_5 246/795  maize Zea mays 88.89 80.16 FUE_5 247/796  maize Zeamays 88.89 80.16 FUE_5 248/797  maize Zea mays 98.41 95.24 FUE_5249/798  maize Zea mays 99.21 99.21 FUE_5 250/799  maize Zea mays 100100 FUE_5 251/800  rice Oryza sativa 86.51 80.16 FUE_5 252/801  riceOryza sativa 86.51 80.16 FUE_5 253/802  rice Oryza sativa 91.27 83.33FUE_5 254/803  rice Oryza sativa 92.06 83.33 FUE_5 255/804  sesameSesamum indicum 85.71 73.81 FUE_5 256/805  sesame Sesamum indicum 85.7174.6 FUE_5 257/806  sorghum Sorghum bicolor 88.1 78.57 FUE_5 258/807 sorghum Sorghum bicolor 97.62 92.06 FUE_5 259/808  sorghum Sorghumbicolor 97.62 92.06 FUE_5 260/809  sugarcane Saccharum officinarum 88.8980.95 FUE_5 261/810  sugarcane Saccharum officinarum 97.62 93.65 FUE_5262/811  tobacco Nicotiana spp. 85.71 70.63 FUE_5 263/812  tobaccoNicotiana spp. 85.71 70.63 FUE_5 264/813  tobacco Nicotiana spp. 85.7170.63 FUE_5 265/814  wheat Triticum spp. 90.48 76.98 FUE_5 266/815 wheat Triticum spp. 91.27 76.98 FUE_5 267/816  wheat Triticum spp. 90.4876.98 FUE_5 268/817  wheat Triticum spp. 91.27 77.78 FUE_5 269/818 wheat Triticum spp. 85.71 79.37 FUE_40 270/819  barley Hordeum vulgare85.89 77.91 FUE_40 271/820  maize Zea mays 85.89 80.37 FUE_40 272/821 maize Zea mays 85.89 80.37 FUE_40 273/822  maize Zea mays 100 100 FUE_40274/823  rice Oryza sativa 85.89 71.17 FUE_40 275/824  sorghum Sorghumbicolor 86.5 73.01 FUE_40 276/825  sorghum Sorghum bicolor 86.5 73.01FUE_40 277/826  sorghum Sorghum bicolor 90.8 86.5 FUE_40 278/827 sorghum Sorghum bicolor 91.41 87.73 FUE_40 279/828  sorghum Sorghumbicolor 91.41 87.73 FUE_7 280/829  maize Zea mays 98.63 98.63 FUE_7281/830  sorghum Sorghum bicolor 87.67 86.3 FUE_9 282/831  maize Zeamays 100 100 FUE_9 283/832  sorghum Sorghum bicolor 94.35 90.4 FUE_10284/833  maize Zea mays 99.14 99.14 FUE_11 285/834  maize Zea mays 96.8394.44 FUE_11 286/835  maize Zea mays 95.24 94.44 FUE_11 287/836  maizeZea mays 97.62 96.03 FUE_11 288/837  maize Zea mays 97.62 96.03 FUE_11289/838  maize Zea mays 97.62 96.03 FUE_11 290/839  maize Zea mays 98.4196.83 FUE_11 291/840  maize Zea mays 96.83 96.83 FUE_11 292/841  maizeZea mays 97.62 97.62 FUE_11 293/842  maize Zea mays 99.21 99.21 FUE_11294/843  sorghum Sorghum bicolor 91.27 85.71 FUE_11 295/844  sorghumSorghum bicolor 91.27 85.71 FUE_11 296/845  sugarcane Saccharumofficinarum 88.1 84.13 FUE_11 297/846  wheat Triticum spp. 96.03 95.24FUE_12 298/847  maize Zea mays 99.2 98.93 FUE_12 299/848  maize Zea mays99.47 99.2 FUE_12 300/849  maize Zea mays 99.73 99.2 FUE_12 301/850 rice Oryza sativa 90.67 80.53 FUE_13 302/851  maize Zea mays 100 100FUE_14 303/852  barley Hordeum vulgare 85.33 77.33 FUE_14 304/853 barley Hordeum vulgare 85.78 79.56 FUE_14 305/854  barley Hordeumvulgare 87.11 80.44 FUE_14 306/855  barley Hordeum vulgare 88.44 81.78FUE_14 307/856  barley Hordeum vulgare 88.44 81.78 FUE_14 308/857  maizeZea mays 85.78 75.11 FUE_14 309/858  maize Zea mays 85.78 75.11 FUE_14310/859  maize Zea mays 86.22 76 FUE_14 311/860  maize Zea mays 86.22 76FUE_14 312/861  maize Zea mays 87.11 78.22 FUE_14 313/862  maize Zeamays 85.33 81.33 FUE_14 314/863  maize Zea mays 92.44 88.44 FUE_14315/864  maize Zea mays 92.44 88.89 FUE_14 316/865  maize Zea mays 92.8988.89 FUE_14 317/866  maize Zea mays 92.89 89.33 FUE_14 318/867  maizeZea mays 93.33 89.33 FUE_14 319/868  maize Zea mays 93.33 89.33 FUE_14320/869  maize Zea mays 94.67 90.22 FUE_14 321/870  maize Zea mays 100100 FUE_14 322/871  maize Zea mays 100 100 FUE_14 323/872  rice Oryzasativa 85.33 73.33 FUE_14 324/873  rice Oryza sativa 85.33 73.33 FUE_14325/874  rice Oryza sativa 85.33 74.22 FUE_14 326/875  rice Oryza sativa85.33 74.22 FUE_14 327/876  rice Oryza sativa 88 77.78 FUE_14 328/877 rice Oryza sativa 88 77.78 FUE_14 329/878  rice Oryza sativa 85.78 79.56FUE_14 330/879  rice Oryza sativa 85.78 79.56 FUE_14 331/880  rice Oryzasativa 86.67 80.89 FUE_14 332/881  rice Oryza sativa 86.67 81.33 FUE_14333/882  rice Oryza sativa 86.67 81.33 FUE_14 334/883  rice Oryza sativa88 81.78 FUE_14 335/884  rice Oryza sativa 87.56 81.78 FUE_14 336/885 rice Oryza sativa 88 81.78 FUE_14 337/886  rice Oryza sativa 87.56 81.78FUE_14 338/887  rice Oryza sativa 87.56 82.22 FUE_14 339/888  rice Oryzasativa 87.56 82.22 FUE_14 340/889  rice Oryza sativa 87.56 82.67 FUE_14341/890  rice Oryza sativa 87.56 82.67 FUE_14 342/891  rice Oryza sativa88 83.11 FUE_14 343/892  rice Oryza sativa 88 83.11 FUE_14 344/893 sorghum Sorghum bicolor 86.22 76.44 FUE_14 345/894  sorghum Sorghumbicolor 86.22 76.44 FUE_14 346/895  sorghum Sorghum bicolor 86.22 77.33FUE_14 347/896  sorghum Sorghum bicolor 86.67 77.78 FUE_14 348/897 sorghum Sorghum bicolor 93.33 89.78 FUE_14 349/898  sorghum Sorghumbicolor 93.78 89.78 FUE_14 350/899  sorghum Sorghum bicolor 93.78 89.78FUE_14 351/900  sorghum Sorghum bicolor 93.78 90.22 FUE_14 352/901 sorghum Sorghum bicolor 93.78 90.22 FUE_14 353/902  sorghum Sorghumbicolor 94.67 90.67 FUE_14 354/903  wheat Triticum spp. 87.11 80.44FUE_14 355/904  wheat Triticum spp. 88.89 80.89 FUE_14 356/905  wheatTriticum spp. 87.56 81.33 FUE_14 357/906  wheat Triticum spp. 87.1181.33 FUE_14 358/907  wheat Triticum spp. 88 81.78 FUE_14 359/908  wheatTriticum spp. 89.33 81.78 FUE_14 360/909  wheat Triticum spp. 90.2283.11 FUE_14 361/910  wheat Triticum spp. 89.33 83.11 FUE_15 362/911 maize Zea mays 100 100 FUE_15 363/912  sorghum Sorghum bicolor 89.3288.26 FUE_16 364/913  maize Zea mays 88.21 83.59 FUE_16 365/914  maizeZea mays 100 100 FUE_16 366/915  sorghum Sorghum bicolor 87.69 83.59FUE_17 367/916  barley Hordeum vulgare 88.84 79.68 FUE_17 368/917 barley Hordeum vulgare 88.84 79.68 FUE_17 369/918  maize Zea mays 94.4288.45 FUE_17 370/919  maize Zea mays 100 100 FUE_17 371/920  maize Zeamays 100 100 FUE_17 372/921  rice Oryza sativa 89.24 78.49 FUE_17373/922  sorghum Sorghum bicolor 96.02 92.03 FUE_17 374/923  wheatTriticum spp. 88.45 79.28 FUE_17 375/924  wheat Triticum spp. 89.6480.08 FUE_30 376/925  maize Zea mays 97.8 97.8 FUE_30 377/926  maize Zeamays 98.31 97.97 FUE_30 378/927  maize Zea mays 99.66 99.66 FUE_30379/928  maize Zea mays 99.83 99.83 FUE_30 380/929  maize Zea mays 99.8399.83 FUE_30 381/930  rice Oryza sativa 87.46 79.15 FUE_30 382/931  riceOryza sativa 87.46 79.15 FUE_30 383/932  sorghum Sorghum bicolor 91.6985.93 FUE_30 384/933  sugarcane Saccharum officinarum 92.03 87.29 FUE_31385/934  maize Zea mays 99.15 98.98 FUE_31 386/935  rice Oryza sativa88.05 81.74 FUE_31 387/936  rice Oryza sativa 88.05 81.74 FUE_31388/937  wheat Triticum spp. 85.15 75.94 FUE_32 389/938  maize Zea mays100 100 FUE_32 390/939  rice Oryza sativa 89.47 85.65 FUE_33 391/940 barley Hordeum vulgare 92.8 88.64 FUE_33 392/941  barley Hordeum vulgare93.63 89.75 FUE_33 393/942  maize Zea mays 99.72 99.72 FUE_33 394/943 maize Zea mays 99.72 99.72 FUE_33 395/944  rice Oryza sativa 93.35 90.3FUE_33 396/945  rice Oryza sativa 93.63 90.58 FUE_33 397/946  sorghumSorghum bicolor 85.32 75.35 FUE_33 398/947  sorghum Sorghum bicolor85.32 75.35 FUE_33 399/948  sorghum Sorghum bicolor 97.51 96.68 FUE_33400/949  sorghum Sorghum bicolor 97.78 96.95 FUE_33 401/950  sugarcaneSaccharum officinarum 85.04 75.9 FUE_33 402/951  sugarcane Saccharumofficinarum 97.51 96.68 FUE_33 403/952  wheat Triticum spp. 93.35 89.75FUE_34_Evo 404/953  maize Zea mays 96.25 94.58 FUE_34_Evo 405/954  maizeZea mays 96.25 94.58 FUE_34_Evo 406/955  rice Oryza sativa 86.25 76.67FUE_34_Evo 407/956  rice Oryza sativa 87.92 78.33 FUE_34_Evo 408/957 rice Oryza sativa 92.5 85.83 FUE_34_Evo 409/958  rice Oryza sativa 92.9286.25 FUE_34_Evo 410/959  wheat Triticum spp. 87.5 78.75 FUE_34_Pat411/960  maize Zea mays 100 100 FUE_34_Pat 412/961  rice Oryza sativa85.83 76.67 FUE_34_Pat 413/962  rice Oryza sativa 87.5 78.33 FUE_34_Pat414/963  rice Oryza sativa 92.08 85.42 FUE_34_Pat 415/964  rice Oryzasativa 92.5 85.83 FUE_34_Pat 416/965  wheat Triticum spp. 87.5 78.75FUE_35 417/966  maize Zea mays 98.41 96.03 FUE_35 418/967  maize Zeamays 100 97.62 FUE_35 419/968  maize Zea mays 100 100 FUE_36 420/969 Arabidopsis Arabidopsis thaliana 87.43 74.86 FUE_36 421/970  ArabidopsisArabidopsis thaliana 87.98 75.41 FUE_36 422/971  Arabidopsis Arabidopsisthaliana 90.16 77.87 FUE_36 423/972  Arabidopsis Arabidopsis thaliana90.16 77.87 FUE_36 424/973  Arabidopsis Arabidopsis thaliana 90.98 80.87FUE_36 425/974  Arabidopsis Arabidopsis thaliana 92.35 82.51 FUE_36426/975  Arabidopsis Arabidopsis thaliana 92.35 82.51 FUE_36 427/976 barley Hordeum vulgare 87.43 79.51 FUE_36 428/977  barley Hordeumvulgare 87.43 79.51 FUE_36 429/978  barley Hordeum vulgare 87.7 80.05FUE_36 430/979  barley Hordeum vulgare 92.35 86.34 FUE_36 431/980 barley Hordeum vulgare 97.27 93.72 FUE_36 432/981  barley Hordeumvulgare 97.27 93.72 FUE_36 433/982  cotton Gossypium ssp. 87.16 77.87FUE_36 434/983  cotton Gossypium ssp. 85.79 78.42 FUE_36 435/984  cottonGossypium ssp. 89.89 82.24 FUE_36 436/985  cotton Gossypium ssp. 90.1682.79 FUE_36 437/986  maize Zea mays 91.8 84.97 FUE_36 438/987  maizeZea mays 98.91 98.91 FUE_36 439/988  maize Zea mays 98.91 98.91 FUE_36440/989  maize Zea mays 100 99.73 FUE_36 441/990  poplar Populus spp91.53 83.06 FUE_36 442/991  poplar Populus spp 91.53 83.33 FUE_36443/992  rice Oryza sativa 90.98 83.06 FUE_36 444/993  rice Oryza sativa90.98 83.06 FUE_36 445/994  rice Oryza sativa 91.26 86.61 FUE_36446/995  rice Oryza sativa 91.26 86.61 FUE_36 447/996  rice Oryza sativa96.17 92.9 FUE_36 448/997  rice Oryza sativa 96.17 92.9 FUE_36 449/998 sorghum Sorghum bicolor 92.62 87.16 FUE_36 450/999  sorghum Sorghumbicolor 99.18 99.18 FUE_36 451/1000 sorghum Sorghum bicolor 99.18 99.18FUE_36 452/1001 soybean Glycine max 87.7 77.87 FUE_36 453/1002 soybeanGlycine max 89.34 78.96 FUE_36 454/1003 soybean Glycine max 89.34 79.51FUE_36 455/1004 soybean Glycine max 89.34 79.51 FUE_36 456/1005 soybeanGlycine max 89.62 79.78 FUE_36 457/1006 soybean Glycine max 92.35 84.15FUE_36 458/1007 sugarcane Saccharum officinarum 98.91 98.63 FUE_36459/1008 sugarcane Saccharum officinarum 98.91 98.91 FUE_36 460/1009sunflower Helianthus annuus 87.43 76.78 FUE_36 461/1010 tobaccoNicotiana spp. 91.53 83.61 FUE_36 462/1011 tomato Solanum lycopersicum87.7 78.69 FUE_36 463/1012 tomato Solanum lycopersicum 87.7 78.69 FUE_36464/1013 tomato Solanum lycopersicum 92.62 84.7 FUE_36 465/1014 wheatTriticum spp. 86.89 80.05 FUE_36 466/1015 wheat Triticum spp. 91.5385.52 FUE_36 467/1016 wheat Triticum spp. 97.27 93.99 FUE_37 468/1017barley Hordeum vulgare 91.55 83.1 FUE_37 469/1018 barley Hordeum vulgare91.55 83.1 FUE_37 470/1019 maize Zea mays 100 100 FUE_37 471/1020 riceOryza sativa 95.07 88.03 FUE_37 472/1021 rice Oryza sativa 95.07 88.03FUE_37 473/1022 sugarcane Saccharum officinarum 97.18 93.66 FUE_37474/1023 wheat Triticum spp. 91.55 83.1 FUE_38 475/1024 ArabidopsisArabidopsis thaliana 86.79 69.97 FUE_38 476/1025 Arabidopsis Arabidopsisthaliana 86.79 69.97 FUE_38 477/1026 Arabidopsis Arabidopsis thaliana86.79 70.27 FUE_38 478/1027 Arabidopsis Arabidopsis thaliana 85.89 73.27FUE_38 479/1028 Arabidopsis Arabidopsis thaliana 85.89 73.27 FUE_38480/1029 Arabidopsis Arabidopsis thaliana 85.89 73.27 FUE_38 481/1030barley Hordeum vulgare 85.29 67.57 FUE_38 482/1031 barley Hordeumvulgare 85.29 67.57 FUE_38 483/1032 barley Hordeum vulgare 85.59 68.17FUE_38 484/1033 barley Hordeum vulgare 85.89 69.37 FUE_38 485/1034barley Hordeum vulgare 85.89 69.37 FUE_38 486/1035 barley Hordeumvulgare 86.19 71.17 FUE_38 487/1036 barley Hordeum vulgare 86.19 71.17FUE_38 488/1037 barley Hordeum vulgare 85.59 73.87 FUE_38 489/1038cotton Gossypium ssp. 85.89 69.97 FUE_38 490/1039 cotton Gossypium ssp.85.29 69.97 FUE_38 491/1040 cotton Gossypium ssp. 85.89 72.97 FUE_38492/1041 cotton Gossypium ssp. 87.09 72.97 FUE_38 493/1042 cottonGossypium ssp. 85.89 73.27 FUE_38 494/1043 cotton Gossypium ssp. 86.7973.87 FUE_38 495/1044 cotton Gossypium ssp. 86.49 74.17 FUE_38 496/1045cotton Gossypium ssp. 89.79 77.48 FUE_38 497/1046 grape Vitis ssp. 86.7972.97 FUE_38 498/1047 grape Vitis ssp. 86.79 72.97 FUE_38 499/1048 grapeVitis ssp. 90.09 80.18 FUE_38 500/1049 maize Zea mays 85.29 67.87 FUE_38501/1050 maize Zea mays 85.29 67.87 FUE_38 502/1051 maize Zea mays 85.2968.47 FUE_38 503/1052 maize Zea mays 85.59 71.17 FUE_38 504/1053 maizeZea mays 85.89 71.17 FUE_38 505/1054 maize Zea mays 85.89 71.17 FUE_38506/1055 maize Zea mays 85.89 71.17 FUE_38 507/1056 maize Zea mays 85.8971.47 FUE_38 508/1057 maize Zea mays 85.89 71.47 FUE_38 509/1058 maizeZea mays 86.19 73.87 FUE_38 510/1059 maize Zea mays 86.19 74.77 FUE_38511/1060 maize Zea mays 86.19 74.77 FUE_38 512/1061 maize Zea mays 100100 FUE_38 513/1062 poplar Populus spp 89.49 79.28 FUE_38 514/1063poplar Populus spp 89.49 79.88 FUE_38 515/1064 rice Oryza sativa 85.5968.17 FUE_38 516/1065 rice Oryza sativa 85.59 68.17 FUE_38 517/1066 riceOryza sativa 85.59 68.77 FUE_38 518/1067 rice Oryza sativa 85.59 68.77FUE_38 519/1068 rice Oryza sativa 87.39 72.07 FUE_38 520/1069 rice Oryzasativa 87.39 72.07 FUE_38 521/1070 rice Oryza sativa 86.79 73.57 FUE_38522/1071 rice Oryza sativa 86.79 73.57 FUE_38 523/1072 rice Oryza sativa85.89 73.87 FUE_38 524/1073 rice Oryza sativa 85.89 73.87 FUE_38525/1074 rice Oryza sativa 86.79 75.38 FUE_38 526/1075 rice Oryza sativa86.79 75.38 FUE_38 527/1076 rice Oryza sativa 96.4 91.59 FUE_38 528/1077rice Oryza sativa 96.4 91.59 FUE_38 529/1078 sorghum Sorghum bicolor85.29 67.87 FUE_38 530/1079 sorghum Sorghum bicolor 85.29 67.87 FUE_38531/1080 sorghum Sorghum bicolor 86.19 69.07 FUE_38 532/1081 sorghumSorghum bicolor 85.89 71.77 FUE_38 533/1082 sorghum Sorghum bicolor87.39 72.07 FUE_38 534/1083 sorghum Sorghum bicolor 99.1 97.6 FUE_38535/1084 sorghum Sorghum bicolor 99.1 97.6 FUE_38 536/1085 soybeanGlycine max 85.29 67.27 FUE_38 537/1086 soybean Glycine max 85.29 67.87FUE_38 538/1087 soybean Glycine max 85.89 69.67 FUE_38 539/1088 soybeanGlycine max 86.49 71.47 FUE_38 540/1089 soybean Glycine max 86.79 71.77FUE_38 541/1090 soybean Glycine max 85.89 72.67 FUE_38 542/1091 soybeanGlycine max 85.29 73.27 FUE_38 543/1092 soybean Glycine max 86.79 74.17FUE_38 544/1093 soybean Glycine max 86.79 74.17 FUE_38 545/1094 soybeanGlycine max 85.59 75.38 FUE_38 546/1095 soybean Glycine max 88.29 77.78FUE_38 547/1096 soybean Glycine max 88.29 78.08 FUE_38 548/1097 soybeanGlycine max 88.89 78.38 FUE_38 549/1098 sugarcane Saccharum officinarum85.29 67.87 FUE_38 550/1099 sugarcane Saccharum officinarum 86.49 72.07FUE_38 551/1100 sugarcane Saccharum officinarum 86.79 75.08 FUE_38552/1101 sugarcane Saccharum officinarum 99.4 97.9 FUE_38 553/1102tobacco Nicotiana spp. 86.79 72.97 FUE_38 554/1103 tobacco Nicotianaspp. 86.79 72.97 FUE_38 555/1104 tobacco Nicotiana spp. 87.39 75.38FUE_38 556/1105 tobacco Nicotiana spp. 87.99 76.28 FUE_38 557/1106tobacco Nicotiana spp. 87.99 76.28 FUE_38 558/1107 tomato Solanumlycopersicum 86.19 69.67 FUE_38 559/1108 tomato Solanum lycopersicum86.49 72.37 FUE_38 560/1109 tomato Solanum lycopersicum 90.69 78.38FUE_38 561/1110 tomato Solanum lycopersicum 90.69 78.38 FUE_38 562/1111wheat Triticum spp. 85.59 68.17 FUE_38 563/1112 wheat Triticum spp.85.89 69.67 FUE_38 564/1113 wheat Triticum spp. 86.19 71.17 FUE_38565/1114 wheat Triticum spp. 85.89 71.47 FUE_38 566/1115 wheat Triticumspp. 85.89 71.47 FUE_38 567/1116 wheat Triticum spp. 86.49 71.77 FUE_38568/1117 wheat Triticum spp. 85.59 73.57 FUE_38 569/1118 wheat Triticumspp. 93.69 86.49 FUE_39 570/1119 Arabidopsis Arabidopsis thaliana 91.9381.17 FUE_39 571/1120 Arabidopsis Arabidopsis thaliana 91.93 81.17FUE_39 572/1121 Arabidopsis Arabidopsis thaliana 91.93 81.17 FUE_39573/1122 Arabidopsis Arabidopsis thaliana 91.48 81.61 FUE_39 574/1123Arabidopsis Arabidopsis thaliana 91.48 81.61 FUE_39 575/1124 canolaBrassica ssp. 90.13 79.82 FUE_39 576/1125 barley Hordeum vulgare 93.2779.82 FUE_39 577/1126 barley Hordeum vulgare 93.27 79.82 FUE_39 578/1127barley Hordeum vulgare 90.58 80.72 FUE_39 579/1128 barley Hordeumvulgare 90.58 80.72 FUE_39 580/1129 barley Hordeum vulgare 98.65 94.62FUE_39 581/1130 barley Hordeum vulgare 98.65 94.62 FUE_39 582/1131cotton Gossypium ssp. 93.27 80.72 FUE_39 583/1132 cotton Gossypium ssp.93.27 81.61 FUE_39 584/1133 cotton Gossypium ssp. 94.17 82.06 FUE_39585/1134 cotton Gossypium ssp. 94.62 82.06 FUE_39 586/1135 cottonGossypium ssp. 94.17 82.06 FUE_39 587/1136 cotton Gossypium ssp. 94.1782.96 FUE_39 588/1137 cotton Gossypium ssp. 95.07 83.86 FUE_39 589/1138grape Vitis ssp. 93.72 82.96 FUE_39 590/1139 grape Vitis ssp. 93.2782.96 FUE_39 591/1140 grape Vitis ssp. 93.72 82.96 FUE_39 592/1141 maizeZea mays 94.62 78.92 FUE_39 593/1142 maize Zea mays 93.27 79.82 FUE_39594/1143 maize Zea mays 92.83 80.27 FUE_39 595/1144 maize Zea mays 93.2780.72 FUE_39 596/1145 maize Zea mays 94.17 80.72 FUE_39 597/1146 maizeZea mays 100 100 FUE_39 598/1147 maize Zea mays 100 100 FUE_39 599/1148peach Prunus persica 93.27 81.17 FUE_39 600/1149 poplar Populus spp93.27 82.06 FUE_39 601/1150 poplar Populus spp 93.27 82.06 FUE_39602/1151 poplar Populus spp 92.38 82.51 FUE_39 603/1152 poplar Populusspp 94.17 82.96 FUE_39 604/1153 poplar Populus spp 94.17 82.96 FUE_39605/1154 rice Oryza sativa 94.62 80.72 FUE_39 606/1155 rice Oryza sativa94.62 81.17 FUE_39 607/1156 rice Oryza sativa 93.72 82.96 FUE_39608/1157 rice Oryza sativa 93.72 82.96 FUE_39 609/1158 rice Oryza sativa95.07 91.93 FUE_39 610/1159 rice Oryza sativa 95.07 91.93 FUE_39611/1160 sorghum Sorghum bicolor 95.52 81.17 FUE_39 612/1161 sorghumSorghum bicolor 95.52 81.17 FUE_39 613/1162 sorghum Sorghum bicolor99.55 98.65 FUE_39 614/1163 soybean Glycine max 93.72 82.51 FUE_39615/1164 soybean Glycine max 93.72 82.51 FUE_39 616/1165 soybean Glycinemax 94.62 83.41 FUE_39 617/1166 soybean Glycine max 94.62 83.41 FUE_39618/1167 sugarcane Saccharum officinarum 94.17 81.61 FUE_39 619/1168sunflower Helianthus annuus 91.03 77.13 FUE_39 620/1169 sunflowerHelianthus annuus 93.27 82.96 FUE_39 621/1170 tobacco Nicotiana spp. 8777.58 FUE_39 622/1171 tobacco Nicotiana spp. 89.69 78.92 FUE_39 623/1172tobacco Nicotiana spp. 92.83 82.51 FUE_39 624/1173 tomato Solanumlycopersicum 89.69 79.82 FUE_39 625/1174 tomato Solanum lycopersicum91.48 81.17 FUE_39 626/1175 tomato Solanum lycopersicum 92.38 82.51FUE_39 627/1176 wheat Triticum spp. 93.27 79.82 FUE_39 628/1177 wheatTriticum spp. 92.83 82.96 FUE_39 629/1178 wheat Triticum spp. 99.1 94.62FUE_41 630/1179 maize Zea mays 98.76 98.76 FUE_41 631/1180 maize Zeamays 100 100 FUE_41 632/1181 sorghum Sorghum bicolor 96.27 92.55 FUE_41633/1182 sorghum Sorghum bicolor 96.89 93.79 FUE_41 634/1183 sorghumSorghum bicolor 96.89 93.79 FUE_43 635/1184 barley Hordeum vulgare 91.2785.71 FUE_43 636/1185 barley Hordeum vulgare 91.27 85.71 FUE_43 637/1186maize Zea mays 100 100 FUE_43 638/1187 rice Oryza sativa 90.48 85.71FUE_43 639/1188 rice Oryza sativa 90.48 85.71 FUE_43 640/1189 sorghumSorghum bicolor 99.21 97.62 FUE_43 641/1190 sorghum Sorghum bicolor99.21 97.62 FUE_43 642/1191 sugarcane Saccharum officinarum 86.51 85.71FUE_43 643/1192 sugarcane Saccharum officinarum 98.41 97.62 FUE_43644/1193 wheat Triticum spp. 91.27 85.71 FUE_44 645/1194 barley Hordeumvulgare 87.72 83.04 FUE_44 646/1195 barley Hordeum vulgare 88.3 84.21FUE_44 647/1196 maize Zea mays 98.83 98.25 FUE_44 648/1197 rice Oryzasativa 90.06 80.7 FUE_44 649/1198 rice Oryza sativa 91.81 85.38 FUE_44650/1199 sorghum Sorghum bicolor 86.55 82.46 FUE_44 651/1200 sorghumSorghum bicolor 96.49 91.81 FUE_44 652/1201 sugarcane Saccharumofficinarum 93.57 86.55 FUE_44 653/1202 sugarcane Saccharum officinarum94.74 87.72 FUE_44 654/1203 sugarcane Saccharum officinarum 97.08 91.81FUE_44 655/1204 sugarcane Saccharum officinarum 97.08 92.4 FUE_44656/1205 wheat Triticum spp. 87.72 83.63 FUE_44 657/1206 wheat Triticumspp. 88.89 84.21 FUE_44 658/1207 wheat Triticum spp. 88.89 84.8 FUE_44659/1208 wheat Triticum spp. 90.64 85.96 FUE_45 660/1209 barley Hordeumvulgare 86.83 78.44 FUE_45 661/1210 barley Hordeum vulgare 86.83 78.44FUE_45 662/1211 maize Zea mays 98.2 94.01 FUE_45 663/1212 maize Zea mays100 100 FUE_45 664/1213 rice Oryza sativa 88.62 80.24 FUE_45 665/1214rice Oryza sativa 88.62 80.24 FUE_45 666/1215 sorghum Sorghum bicolor100 98.2 FUE_45 667/1216 sugarcane Saccharum officinarum 100 98.2 FUE_45668/1217 wheat Triticum spp. 85.03 76.65 FUE_45 669/1218 wheat Triticumspp. 85.03 77.25 FUE_45 670/1219 wheat Triticum spp. 85.03 77.25 FUE_45671/1220 wheat Triticum spp. 85.63 77.84 FUE_45 672/1221 wheat Triticumspp. 97.01 95.21 FUE_46 673/1222 maize Zea mays 100 100 FUE_47 674/1223maize Zea mays 89.72 85.77 FUE_47 675/1224 maize Zea mays 100 100 FUE_47676/1225 maize Zea mays 100 100 FUE_47 677/1226 maize Zea mays 100 100FUE_47 678/1227 sugarcane Saccharum officinarum 88.14 86.17 FUE_48679/1228 barley Hordeum vulgare 89.29 81.75 FUE_48 680/1229 maize Zeamays 86.24 79.76 FUE_48 681/1230 maize Zea mays 99.21 99.07 FUE_48682/1231 maize Zea mays 99.34 99.21 FUE_48 683/1232 rice Oryza sativa86.64 79.23 FUE_48 684/1233 rice Oryza sativa 86.64 79.23 FUE_48685/1234 rice Oryza sativa 86.64 79.23 FUE_48 686/1235 rice Oryza sativa90.34 84.26 FUE_48 687/1236 rice Oryza sativa 90.34 84.26 FUE_48688/1237 sorghum Sorghum bicolor 94.05 90.61 FUE_48 689/1238 sorghumSorghum bicolor 94.05 90.61 FUE_48 690/1239 wheat Triticum spp. 89.6881.35 FUE_48 691/1240 wheat Triticum spp. 89.42 81.61 FUE_49 692/1241maize Zea mays 96.03 92.82 FUE_49 693/1242 maize Zea mays 98.11 97.35FUE_49 694/1243 maize Zea mays 99.24 98.68 FUE_49 695/1244 rice Oryzasativa 89.04 81.1 FUE_49 696/1245 rice Oryza sativa 89.04 81.1 FUE_49697/1246 wheat Triticum spp. 86.96 77.13 FUE_49 698/1247 wheat Triticumspp. 87.33 77.32 FUE_50 699/1248 maize Zea mays 99.73 99.73 FUE_50700/1249 sugarcane Saccharum officinarum 91.42 88.2 FUE_50 701/1250sugarcane Saccharum officinarum 92.49 89.28 FUE_51 702/1251 barleyHordeum vulgare 91.5 84.75 FUE_51 703/1252 barley Hordeum vulgare 91.584.75 FUE_51 704/1253 barley Hordeum vulgare 94.12 88.45 FUE_51 705/1254maize Zea mays 100 100 FUE_51 706/1255 rice Oryza sativa 92.59 85.4FUE_51 707/1256 rice Oryza sativa 92.59 85.4 FUE_51 708/1257 rice Oryzasativa 95.86 93.46 FUE_51 709/1258 rice Oryza sativa 96.08 93.68 FUE_51710/1259 sorghum Sorghum bicolor 92.16 83.22 FUE_51 711/1260 sorghumSorghum bicolor 92.16 83.22 FUE_51 712/1261 sugarcane Saccharumofficinarum 92.16 83.88 FUE_51 713/1262 sugarcane Saccharum officinarum98.91 98.26 FUE_51 714/1263 wheat Triticum spp. 91.29 84.97 FUE_51715/1264 wheat Triticum spp. 94.77 88.89 FUE_52 716/1265 barley Hordeumvulgare 89.35 83.8 FUE_52 717/1266 barley Hordeum vulgare 89.35 83.8FUE_52 718/1267 maize Zea mays 100 99.77 FUE_52 719/1268 maize Zea mays100 99.77 FUE_52 720/1269 rice Oryza sativa 90.05 85.88 FUE_52 721/1270rice Oryza sativa 90.05 86.11 FUE_52 722/1271 rice Oryza sativa 90.0586.11 FUE_52 723/1272 sorghum Sorghum bicolor 93.06 90.97 FUE_52724/1273 sugarcane Saccharum officinarum 94.68 92.82 FUE_52 725/1274wheat Triticum spp. 89.35 83.33 FUE_53 726/1275 barley Hordeum vulgare86.86 73.73 FUE_53 727/1276 maize Zea mays 95.98 94.37 FUE_53 728/1277maize Zea mays 100 100 FUE_53 729/1278 rice Oryza sativa 93.57 86.86FUE_53 730/1279 rice Oryza sativa 93.57 86.86 FUE_53 731/1280 wheatTriticum spp. 89.54 80.43 FUE_54 732/1281 maize Zea mays 90.94 88.77FUE_54 733/1282 maize Zea mays 96.74 96.74 FUE_54 734/1283 sorghumSorghum bicolor 87.32 83.7 FUE_54 735/1284 sorghum Sorghum bicolor 87.3283.7 FUE_54 736/1285 sorghum Sorghum bicolor 90.22 86.59 FUE_54 737/1286sugarcane Saccharum officinarum 90.94 87.32 FUE_54 738/1287 sugarcaneSaccharum officinarum 90.94 87.32 FUE_55 739/1288 maize Zea mays 92.3189.62 FUE_100 740/1289 barley Hordeum vulgare 88.89 81.77 FUE_100741/1290 barley Hordeum vulgare 88.89 81.77 FUE_100 742/1291 maize Zeamays 85.19 74.64 FUE_100 743/1292 maize Zea mays 85.19 74.64 FUE_100744/1293 maize Zea mays 98.29 98.01 FUE_100 745/1294 maize Zea mays99.72 99.72 FUE_100 746/1295 rice Oryza sativa 86.61 78.92 FUE_100747/1296 rice Oryza sativa 86.61 78.92 FUE_100 748/1297 sorghum Sorghumbicolor 85.19 74.93 FUE_100 749/1298 sorghum Sorghum bicolor 87.46 78.06FUE_100 750/1299 sorghum Sorghum bicolor 87.46 78.06 FUE_100 751/1300sorghum Sorghum bicolor 92.02 88.03 FUE_100 752/1301 sugarcane Saccharumofficinarum 89.17 78.63 FUE_100 753/1302 sugarcane Saccharum officinarum91.45 87.75 FUE_100 754/1303 wheat Triticum spp. 87.18 79.77 FUE_101755/1304 maize Zea mays 100 100 FUE_101 756/1305 rice Oryza sativa 91.7684.82 FUE_101 757/1306 rice Oryza sativa 91.76 84.82 FUE_102 758/1307maize Zea mays 87.2 83.65 FUE_102 759/1308 maize Zea mays 94.31 93.84FUE_102 760/1309 maize Zea mays 96.21 96.21 FUE_501 761/1310 ArabidopsisArabidopsis thaliana 87.13 75.86 FUE_501 762/1311 ArabidopsisArabidopsis thaliana 87.59 76.55 FUE_501 763/1312 ArabidopsisArabidopsis thaliana 100 100 FUE_502 764/1313 Arabidopsis Arabidopsisthaliana 100 100 FUE_503 765/1314 Arabidopsis Arabidopsis thaliana 100100 FUE_504 766/1315 Arabidopsis Arabidopsis thaliana 100 100 FUE_505767/1316 Arabidopsis Arabidopsis thaliana 100 100

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications and GenBank Accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application or GenBank Accession numberwas specifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

What is claimed is:
 1. A method of increasing biomass and/or growth rateof a plant under nitrogen limiting conditions of 0.01-3 mM nitrogen,comprising: (a) over-expressing within a whole plant an exogenouspolynucleotide comprising a nucleic acid sequence encoding thepolypeptide set forth by SEQ ID NO:173, (b) selecting said whole plantexpressing said exogenous polynucleotide for an increased biomass and/orgrowth rate under limiting-nitrogen conditions of 0.01-3 mM nitrogenwhen grown on soil as compared to a whole control plant which is grownunder the same growth conditions, and (c) obtaining a progeny of saidwhole plant over-expressing said exogenous polynucleotide and selectedaccording to step (b), thereby increasing the biomass and/or growth rateof the plant under the nitrogen limiting conditions.
 2. The method ofclaim 1, wherein said exogenous polynucleotide comprises the nucleicacid sequence set forth by SEQ ID NO: 64 or a codon-optimized sequenceof said nucleic acid sequence for expression in the plant.
 3. The methodof claim 1, further comprising growing said whole plant or said progenyexpressing said exogenous polynucleotide under the nitrogen limitingconditions.
 4. A method of producing seeds of a crop, comprising: (a)selecting a parent plant transformed with an exogenous polynucleotidecomprising a nucleic acid sequence encoding the polypeptide set forth bySEQ ID NO:173 for an increased biomass and/or growth rate under nitrogenlimiting conditions of 0.01-3 mM nitrogen when grown on soil as comparedto a control plant which is grown under the same growth conditions, (b)growing on soil a seed-producing whole plant from said parent plantresultant of step (a), wherein said seed-producing whole plant whichcomprises said exogenous polynucleotide has said increased biomassand/or growth rate under nitrogen limiting conditions, and (c) producingseeds from said seed-producing whole plant resultant of step (b),thereby producing seeds of the crop.
 5. The method of claim 4, whereinsaid exogenous polynucleotide comprises the nucleic acid sequence setforth by SEQ ID NO: 64 or a codon-optimized sequence of said nucleicacid sequence for expression in the plant.
 6. The method of claim 4,wherein said exogenous polynucleotide comprises the nucleic acidsequence set forth by SEQ ID NO:
 64. 7. The method of claim 1, whereinsaid exogenous polynucleotide comprises the nucleic acid sequence setforth by SEQ ID NO:
 64. 8. A method of increasing biomass and/or growthrate of a regenerated plant under nitrogen limiting conditions of0.01-0.05 mM nitrogen, comprising: (a) over-expressing within a wholeplant an exogenous polynucleotide comprising a nucleic acid sequenceencoding the polypeptide set forth by SEQ ID NO:173, (b) selecting saidwhole plant over-expressing said exogenous polynucleotide for anincreased biomass and/or growth rate under nitrogen limiting conditionsof 0.01-0.05 mM nitrogen when grown on soil as compared to a controlwhole plant which is grown under the same growth conditions, and (c)obtaining a progeny of said whole plant over-expressing said exogenouspolynucleotide and selected according to step (b), thereby increasingthe biomass and/or growth rate under nitrogen limiting deficiencyconditions of the regenerated plant.
 9. A method of producing seeds of acrop, comprising: (a) selecting a parent plant transformed with anexogenous polynucleotide comprising a nucleic acid sequence encoding thepolypeptide set forth by SEQ ID NO:173 for an increased biomass and/orgrowth rate under nitrogen limiting deficiency conditions of 0.01-0.05mM nitrogen when grown on soil as compared to a control plant which isgrown under the same growth conditions, (b) growing a seed-producingwhole plant from said parent plant resultant of step (a), wherein saidseed-producing whole plant which comprises said exogenous polynucleotidehas said increased biomass and/or growth rate under said nitrogenlimiting conditions, and (c) producing seeds from said seed-producingwhole plant resultant of step (b), thereby producing seeds of the crop.10. The method of claim 1, wherein said expression of said exogenouspolynucleotide is controlled by a constitutive promoter.
 11. The methodof claim 4, wherein said expression of said exogenous polynucleotide iscontrolled by a constitutive promoter.
 12. The method of claim 8,wherein said expression of said exogenous polynucleotide is controlledby a constitutive promoter.
 13. The method of claim 9, wherein saidexpression of said exogenous polynucleotide is controlled by aconstitutive promoter.